Methods and compositions for the treatment of motor neuron injury and neuropathy

ABSTRACT

Disclosed are therapeutic treatment methods, compositions and devices for maintaining neural pathways in a mammal, including enhancing survival of neurons at risk of dying, inducing cellular repair of damaged neurons and neural pathways, and stimulating neurons to maintain their differentiated phenotype. In one embodiment, the invention provides means for stimulating CAM expression in neurons. The invention also provides means for evaluating the status of nerve tissue, including means for detecting and monitoring neuropathies in a mammal. The methods, devices and compositions include a morphogen or morphogen-stimulating agent provided to the mammal in a therapeutically effective concentration.

RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of U.S. Pat.No. 08/260,675, filed Jun. 16, 1994, which is a file wrappercontinuation of U.S. Pat. No. 08/126,100, filed Sep. 23, 1993, which isa file wrapper continuation of U.S. Pat. No. 07/922,813, filed Jul. 31,1992 filed as a continuation-in-part of U.S. Pat. No. 07/752,764 andcopending U.S. Pat. No. 07/753,059, both filed Aug. 30, 1991 ascontinuations-in-part of U.S. Pat. No. 07/667,274, filed Mar. 11, 1991.The above-mentioned applications are incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] The mammalian nervous system comprises a peripheral nervoussystem (PNS) and a central nervous system (CNS, comprising the brain andspinal cord), and is composed of two principal classes of cells: neuronsand glial cells. The glial cells fill the spaces between neurons,nourishing them and modulating their function. Certain glial cells, suchas Schwann cells in the PNS and oligodendrocytes in the CNS, alsoprovide a myelin sheath that surrounds neural processes. The myelinsheath enables rapid conduction along the neuron. In the peripheralnervous system, axons of multiple neurons may bundle together in orderto form a nerve fiber. These, in turn, may be combined into fascicles orbundles.

[0003] During development, differentiating neurons from the central andperipheral nervous systems send out axons that grow and make contactwith specific target cells. In some cases, axons must cover enormousdistances; some grow into the periphery, whereas others are confinedwithin the central nervous system. In mammals, this stage ofneurogenesis is complete during the embryonic phase of life and neuronalcells do not multiply once they have fully differentiated.

[0004] A host of neuropathies have been identified that affect thenervous system. The neuropathies, which may affect neurons themselves orassociated glial cells, may result from cellular metabolic dysfunction,infection, exposure to toxic agents, autoimmunity, malnutrition, orischemia. In some cases, the cellular neuropathy is thought to inducecell death directly. In other cases, the neuropathy may inducesufficient tissue necrosis to stimulate the body's immune/inflammatorysystem and the immune response to the initial injury then destroysneural pathways.

[0005] Where the damaged neural pathway results from CNS axonal damage,autologous peripheral nerve grafts have been used to bridge lesions inthe central nervous system and to allow axons to make it back to theirnormal target area. In contrast to CNS neurons, neurons of theperipheral nervous system can extend new peripheral processes inresponse to axonal damage. This regenerative property of peripheralnervous system axons is thought to be sufficient to allow grafting ofthese segments to CNS axons. Successful grafting appears to be limited,however, by a number of factors, including the length of the CNS axonallesion to be bypassed, and the distance of the graft sites from the CNSneuronal cell bodies, with successful grafts occurring near the cellbody.

[0006] Within the peripheral nervous system, this cellular regenerativeproperty of neurons has limited ability to repair function to a damagedneural pathway. Specifically, the new axons extend randomly, and areoften misdirected, making contact with inappropriate targets that cancause abnormal function. For example, if a motor nerve is damaged,regrowing axons may contact the wrong muscles, resulting in paralysis.In addition, where severed nerve processes result in a gap of longerthan a few millimeters, e.g., greater than 10 millimeters (mm),appropriate nerve regeneration does not occur, either because theprocesses fail to grow the necessary distance, or because of misdirectedaxonal growth. Efforts to repair peripheral nerve damage by surgicalmeans has met with mixed results, particularly where damage extends overa significant distance. In some cases, the suturing steps used to obtainproper alignment of severed nerve ends stimulates the formulation ofscar tissue which is thought to inhibit axon regeneration. Even wherescar tissue formation has been reduced, as with the use of nerveguidance channels or other tubular prostheses, successful regenerationgenerally still is limited to nerve damage of less than 10 millimetersin distance. In addition, the reparative ability of peripheral neuronsis significantly inhibited where an injury or neuropathy affects thecell body itself or results in extensive degeneration of a distal axon.

[0007] Mammalian neural pathways also are at risk due to damage causedby neoplastic lesions. Neoplasias of both the neurons and glial cellshave been identified. Transformed cells of neural origin generally losetheir ability to behave as normal differentiated cells and can destroyneural pathways by loss of function. In addition, the proliferatingtumors may induce lesions by distorting normal nerve tissue structure,inhibiting pathways by compressing nerves, inhibiting cerbrospinal fluidor blood supply flow, and/or by stimulating the body's immune response.Metastatic tumors, which are a significant cause of neoplastic lesionsin the brain and spinal cord, also similarly may damage neural pathwaysand induce neuronal cell death.

[0008] One type of morphoregulatory molecule associated with neuronalcell growth, differentiation and development is the cell adhesionmolecule (“CAM”), most notably the nerve cell adhesion molecule (N-CAM).The CAMs are members the immunoglobulin super-family. They mediatecell-cell interactions in developing and adult tissues throughhomophilic binding, i.e., CAM-CAM binding on apposing cells. A number ofdifferent CAMs have been identified. Of these, the most thoroughlystudied are N-CAM and L-CAM (liver cell adhesion molecules), both ofwhich have been identified on all cells at early stages of development,as well as in different adult tissues. In neural tissue development,N-CAM expression is believed to be important in tissue organization,neuronal migration, nerve-muscle tissue adhesion, retinal formation,synaptogenesis, and neural degeneration. Reduced N-CAM expression alsois thought to be associated with nerve dysfunction. For example,expression of at least one form of N-CAM, N-CAM-1 80, is reduced in amouse demyelinating mutant. Bhat, Brain Res. 452: 373-377 (1988).Reduced levels of N-CAM also have been associated with normal pressurehydrocephalus, Werdelin, Acta Neurol. Scand. 79: 177-181 (1989), andwith type II schizophrenia. Lyons, et al., Biol. Psychiatry 23: 769-775(1988). In addition, antibodies against N-CAM have been shown to disruptfunctional recovery in injured nerves. Remsen, Exp. Neurobiol. 110:268-273 (1990).

[0009] Currently no satisfactory method exists to repair the damagecaused by traumatic injuries of motor neurons and diseases of motorneurons.

[0010] There are 15,000 to 18,000 new cases of spinal cord injury eachyear in the United States. In addition, there are approximately 200,000survivors of spinal cord injury. The annual cost of care for thesepatients exceeds $7 billion. The pathophysiology following acute spinalcord trauma is a complex and not fully understood mechanism. The primarytissue damage caused by mechanical trauma occurs immediately and isirreversible. Allen, J. Am. Med. Assoc. 57: 878-880 (1911). Experimentalevidence indicates that much of the post-traumatic tissue damage is theresult of a reactive process that begins within minutes after the injuryand continues for days or weeks. Janssen, et al., Spine 14: 23-32 (1989)and Panter, et al., (1992). This progressive, self-destructive processincludes pathophysiological mechanisms such as hemorrhage,post-traumatic ischemia, edema, axonal and neuronal necrosis, anddemyelinization followed by cyst formation and infarction. For review,see Tator, et al., J. Neurosurg, 75: 15-26 (1991) and Faden, Crit. Rev.Neurobiol. 7: 175-186 (1993). Proposed injurious factors includeelectrolyte changes whereby increased intracellular calcium initiates acascade of events (Young, J. Neurotrauma 9, Suppl. 1: S9-S25 (1992) andYoung, J. Emerg. Med 11: 13-22 (1993)), biochemical changes withuncontrolled transmitter release (Liu, et al., Cell 66: 807-815 (1991)and Yanase, et al., J. Neurosurg 83: 884-888 (1995), arachidonic acidrelease, free-radical production, lipid peroxidation (Braughler, et al.,J. Neurotrauma 9, Suppl. 1: S1-S7 (1992), eicosanoid production(Demediuk, et al., J. Neurosci. Res. 20: 115-121 (1988), endogenousopioids (Faden, et al., Ann Neurol. 17: 386-390 (1985), metabolicchanges including alterations in oxygen and glucose (Faden, Crit. Rev.Neurobiol. 7: 175-186 (1993)), inflammatory changes (Blight, J.Neurotrauma 9, Suppl. 1: S83-S91 (1992), and astrocytic edema(Kimelberg, J. Neurotrauma 9, Suppl. 1: S71-S81 (1992). For the past 400years surgical approaches including laminectomy and decompression,accompanied by fusion, have been the most commonly practiced treatmentstrategies. Hansebout, “Early Management of Acute Spinal Cord Injury”,pp. 181-196 (1982) and Janssen, et al., Spine 14: 23-32 (1989). However,these procedures have not involved the application of techniques toaugment the regenerative properties of spinal cord tissue.

[0011] A host of diseases of motor neurons have been identified,including demyelinating diseases, myelopathies, and diseases of motorneurons such as amyotrophic lateral sclerosis (ALS). INTERNAL MEDICINE,ch. 121-123 (4th ed., J. H. Stein, ed., Mosby, 1994). Multiple sclerosis(MS) is the most common demyelinating disorder of the central nervoussystem, causing patches of sclerosis (i.e., plaques) in the brain andspinal cord. MS has protean clinical manifestations, depending upon thelocation and size of the plaque. Typical symptoms include visual loss,diplopia, nystagmus, dysarthria, weakness, paresthesias, bladderabnormalities, and mood alterations. Myriad treatments have beenproposed for this long-term variable illness. The list of proposedtreatments encompasses everything from diet to electrical stimulation toacupuncture, emotional support, and various forms of immunosupressivetherapy. None have proved to be satisfactory.

[0012] Progressive loss of lower and upper motor neurons occurs inseveral diseases (e.g., primary lateral sclerosis, spinal muscularatrophy, benign focal amyotrophy). However, ALS is the most common formof motor neuron disease. Loss of both lower and upper motor neuronsoccur in ALS. Symptoms include progressive skeletal muscle wasting,weakness, gasciculations, and cramping. Some cases have predominantinvolvement of brainstem motoneurons (progressive bulbar palsy).Unfortunately, treatment of motor neuron and related diseas is largelysupportive at this time. INTERNAL MEDICINE, ch. 123 (4th ed., J. H.Stein, ed., Mosby, 1994).

[0013] Accordingly, there is a need in the art for treatments of motorneurons disorders and injuries, and related deficits in neuralfunctions.

SUMMARY OF THE INVENTION

[0014] The present invention provides methods and compositions formaintaining neural pathways in a mammal in vivo, including methods forenhancing the survival of neural cells.

[0015] In a preferred embodiment, methods of the invention for treatingmotor neuron defects, including amyotrophic lateral sclerosis, multiplesclerosis, and spinal cord injury comprise administering a morphogencomprising a dimeric protein having an amino acid sequence selected fromthe group consisting of a sequence have 70% homology with the C-terminalseven-cysteine skeleton of human OP-1 (amino acids 330-341 of SEQ IDNO:2), a sequence having greater than 60% amino acid sequence identitywith human OP-1; generic sequence 7 (SEQ ID NO:4); generic sequence 8(SEQ ID NO:6); generic sequence 10 (SEQ ID NO:7); and OPX (SEQ ID NO:3);wherein the morphogen stimulates production of N-CAM or L1 isoforms byan NG108-15 cell in vivo. Spinal cord injuries include injuriesresulting from a tumor, mechanical trauma, and chemical trauma. The sameor similar methods are contemplated to restore motor function in amammal having amyotrophic lateral sclerosis, multiple sclerosis, or aspinal cord injury. Administering one of the aforementioned morphogensalso provides a prophylactic function. Such administration has theeffect of preserving motor function in a mammal having, or at risk ofhaving, amyotrophic lateral sclerosis, multiple sclerosis, or a spinalcord injury. Also according to the invention, morphogen administrationpreserves the integrity of the nigrostriatal pathway.

[0016] Specifically, methods of the invention for treating (pre- orpost-symptomatically) amyotrophic lateral sclerosis, multiple sclerosis,or a spinal cord injury comprise administering a morphogen selected fromthe group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2,60A, GDF-1, BMP2A, BMP2B, DPP, Vg1, Vgr-1, BMP3, BMP5, and BMP6. Suchmorphogens are capable of stimulating production of N-CAM or L1 isoformby an NG108-15 cell in vivo.

[0017] In a particularly-preferred embodiment, the morphogen is asoluble complex, comprising at least one morphogen pro domain, orfragment thereof, non-covalently attached to a mature morphogen.

[0018] In one aspect, the invention features compositions andtherapeutic treatment methods comprising administering to a mammal atherapeutically effective amount of a morphogenic protein (“morphogen”),as defined herein, upon injury to a neural pathway, or in anticipationof such injury, for a time and at a concentration sufficient to maintainthe neural pathway, including repairing damaged pathways, or inhibitingadditional damage thereto.

[0019] In another aspect, the invention features compositions andtherapeutic treatment methods for maintaining neural pathways. Suchtreatment methods include administering to the mammal, upon injury to aneural pathway or in anticipation of such injury, a compound thatstimulates a therapeutically effective concentration of an endogenousmorphogen. These compounds are referred to herein asmorphogen-stimulating agents, and are understood to include substanceswhich, when administered to a mammal, act on tissue(s) or organ(s) thatnormally are responsible for, or capable of, producing a morphogenand/or secreting a morphogen, and which cause endogenous level of themorphogen to be altered.

[0020] In particular, the invention provides methods for protectingneurons from the tissue destructive effects associated with the body'simmune and inflammatory response to nerve injury. The invention alsoprovides methods for stimulating neurons to maintain theirdifferentiated phenotype, including inducing the redifferentiation oftransformed cells of neuronal origin to a morphology characteristic ofuntransformed neurons. In one embodiment, the invention provides meansfor stimulating production of cell adhesion molecules, particularlynerve cell adhesion molecules (N-CAM). The invention also providesmethods, compositions and devices for stimulating cellular repair ofdamaged neurons and neural pathways, including regenerating damageddendrites or axons. In addition, the invention also provides means forevaluating the status of nerve tissue, and for detecting and monitoringneuropathies by monitoring fluctuations in morphogen levels.

[0021] In one aspect of the invention, the morphogens described hereinare useful in repairing damaged neural pathways of the peripheralnervous system. In particular, morphogens are useful for repairingdamaged neural pathways, including transected or otherwise damaged nervefibers. Specifically, the morphogens described herein are capable ofstimulating complete axonal nerve regeneration, includingvascularization and reformation of the myelin sheath. Preferably, themorphogen preferably is provided to the site of injury in abiocompatible, bioresorbable carrier capable of maintaining themorphogen at the site and, where necessary, means for directing axonalgrowth from the proximal to the distal ends of a severed neuron. Forexample, means for directing axonal growth may be required where nerveregeneration is to be induced over an extended distance, such as greaterthan 10 mm. Many carriers capable of providing these functions areenvisioned. For example, useful carriers include substantially insolublematerials or viscous solutions prepared as disclosed herein comprisinglaminin, hyaluronic acid or collagen, or other suitable synthetic,biocompatible polymeric materials such as polylactic, polyglycolic orpolybutyric acids and/or copolymers thereof. A preferred carriercomprises an extracellular matrix composition derived, for example, frommouse sarcoma cells.

[0022] In a particularly preferred embodiment, a morphogen is disposedin a nerve guidance channel which spans the distance of the damagedpathway. The channel acts both as a protective covering and a physicalmeans for guiding growth of a neurite. Useful channels comprise abiocompatible membrane, which may be tubular in structure, having adimension sufficient to span the gap in the nerve to be repaired, andhaving openings adapted to receive severed nerve ends. The membrane maybe made of any biocompatible, nonirritating material, such as siliconeor a biocompatible polymer, such as polyethylene or polyethylene vinylacetate. The casing also may be composed of biocompatible, bioresorbablepolymers, including, for example, collagen, hyaluronic acid, polylactic,polybutyric, and polyglycolic acids. In a preferred embodiment, theouter surface of the channel is substantially impermeable.

[0023] The morphogen may be disposed in the channel in association witha biocompatible carrier material, or it may be adsorbed to or otherwiseassociated with the inner surface of the casing, such as is described inU.S. Pat. No. 5,011,486, provided that the morphogen is accessible tothe severed nerve ends.

[0024] Morphogens described herein are also useful in autologousperipheral nerve segment implants, such as in the repair of damaged ordetached retinas, or other damage to the optic nerve.

[0025] In another aspect of the invention, morphogens described hereinare useful to protect against damage associated with the body'simmune/inflammatory response to an initial injury to nerve tissue. Sucha response may follow trauma to nerve tissue, caused, for example, by anautoimmune dysfunction, neoplastic lesion, infection, chemical ormechanical trauma, disease, by interruption of blood flow to the neuronsor glial cells, or by other trauma to the nerve or surrounding material.For example, the primary damage resulting from hypoxia orischemia-reperfusion following occlusion of a neural blood supply, as inan embolic stroke, is believed to be immunologically associated. Inaddition, at least part of the damage associated with a number ofprimary brain tumors also appears to be immunologically related.Application of a morphogen, either directly or systemically alleviateand/or inhibit the immunologically related response to a neural injury.Alternatively, administration of an agent capable of stimulatingmorphogen expression and/or secretion in vivo, preferably at the site ofinjury, may also be used. Where the injury is to be induced, as duringsurgery or other aggressive clinical treatment, the morphogen or agentmay be provided prior to induction of the injury to provide aneuroprotective effect to the nerve tissue at risk.

[0026] Generally, morphogens useful in methods and compositions of theinvention are dimeric proteins that induce morphogenesis of one or moreeukaryotic (e.g., mammalian) cells, tissues or organs. Tissuemorphogenesis includes de novo or regenerative tissue formation, such asoccurs in a vertebrate embryo during development. Of particular interestare morphogens that induce tissue-specific morphogenesis at least ofbone or neural tissue. As defined herein, a morphogen comprises a pairof polypeptides that, when folded, form a dimeric protein that elicitsmorphogenetic responses in cells and tissues displayingmorphogen-specific receptors. That is, the morphogens generally induce acascade of events including all of the following in a morphogenicallypermissive environment: stimulating proliferation of progenitor cells;stimulating the differentiation of progenitor cells; stimulating theproliferation of differentiated cells; and, supporting the growth andmaintenance of differentiated cells. “Progenitor” cells are uncommittedcells that are competent to differentiate into one or more specifictypes of differentiated cells, depending on their genomic repertoire andthe tissue specificity of the permissive environment in whichmorphogenesis is induced. An exemplary progenitor cell is ahematopoeitic stem cell, a mesenchymal stem cell, a basement epitheliumcell, a neural crest cell, or the like. Further, morphogens can delay ormitigate the onset of senescence- or quiescence-associated loss ofphenotype and/or tissue function. Still further, morphogens canstimulate phenotypic expression of a differentiated cell type, includingexpression of metabolic and/or functional, e.g., secretory, propertiesthereof. In addition, morphogens can induce redifferentiation ofcommitted cells (e.g., osteoblasts, neuroblasts, or the like) underappropriate conditions. As noted above, morphogens that induceproliferation and/or differentiation at least of bone or neural tissue,and/or support the growth, maintenance and/or functional properties ofneural tissue, are of particular interest herein. See, e.g., WO92/15323, WO 93/04692, WO 94/03200 (providing more detailed disclosuresas to the tissue morphogenic properties of these proteins).

[0027] As used herein, the terms “morphogen,” “bone morphogen,” “bonemorphogenic protein,” “BMP,” “morphogenic protein” and “morphogeneticprotein” all embrace the class of proteins typified by human osteogenicprotein 1 (hOP-1). Nucleotide and amino acid sequences for hOP-1 areprovided in SEQ ID NOS:1 and 2, respectively. For ease of description,hOP-1 is considered a representative morphogen. It will be appreciatedthat OP-1 is merely representative of the TGF-β subclass of true tissuemorphogens and is not intended to limit the description. Other known anduseful morphogens include, but are not limited to, BMP-2, BMP-3, BMP-3b,BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13,BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10,GDF-11, GDF-12, 60A, NODAL, UNIVIN, SCREW, ADMP, and NEURAL, andmorphogenically-active amino acid variants of any thereof.

[0028] In specific embodiments, useful morphogens include those sharingthe conserved seven cysteine skeleton, and sharing at least 70% aminoacid sequence homology (similarity), within the C-terminalseven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO:2(hereinafter referred to as the “reference sequence”). In anotherembodiment, the invention encompasses use of biologically active species(phylogenetic) variants of any of the morphogenic proteins recitedherein, including conservative amino acid sequence variants, proteinsencoded by degenerate nucleotide sequence variants, andmorphogenically-active proteins sharing the conserved seven cysteineskeleton as defined herein and encoded by a DNA competent to hybridizeunder standard stringency conditions to a DNA encoding a morphogenicprotein disclosed herein, including, without limitation, OP-1 or BMP-2or BMP-4. Presently, however, the reference sequence is that of residues330-431 of SEQ ID NO:2 (OP-1).

[0029] In still another embodiment, morphogens useful in methods andcompositions of the invention are defined as morphogenically-activeproteins having any one of the generic sequences defined herein,including OPX (SEQ ID NO:3) and Generic Sequences 7 and 8 (SEQ ID NOS:4and 5, respectively), or Generic Sequences 9 and 10 (SEQ ID NOS:6 and 7,respectively). OPX encompasses the observed variation between the knownphylogenetic counterparts of the osteogenic OP-1 and OP-2 proteins, andis described by the amino acid sequence presented herein below and inSEQ ID NO:3. Generic Sequence 9 is a 96 amino acid sequence containingthe C-terminal six cysteine skeleton observed in hOP-1 (residues 335-431of SEQ ID NO:2) and wherein the remaining residues encompass theobserved variation among OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5,BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-15, GDF-1, GDF-3, GDF-5, GDF-6,GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, 60A, UNIVIN, NODAL, DORSALIN,NEURAL, SCREW and ADMP. That is, each of the non-cysteine residues isindependently selected from the corresponding residue in this recitedgroup of known, naturally-sourced proteins. Generic Sequence 10 is a 102amino acid sequence which includes a five amino acid sequence added tothe N-terminus of the Generic Sequence 9 and defines the seven cysteineskeleton observed in hOP-1 (330-431 SEQ ID NO:2). Generic Sequences 7and 8 are 96 and 102 amino acid sequences, respectively, containingeither the six cysteine skeleton (Generic Sequence 7) or the sevencysteine skeleton (Generic Sequence 8) defined by hOP-1 and wherein theremaining non-cysteine residues encompass the observed variation amongOP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, 60A, DPP, Vg1, BMP-5, BMP-6,Vgr-1, and GDF-1.

[0030] Of particular interest are morphogens which, when provided to aspecific tissue of a mammal, induce tissue-specific morphogenesis ormaintain the normal state of differentiation and growth of that tissue.In preferred embodiments, the present morphogens induce the formation ofvertebrate (e.g., avian or mammalian) body tissues, such as but notlimited to nerve, eye, bone, cartilage, bone marrow, ligament, toothdentin, periodontium, liver, kidney, lung, heart, or gastrointestinallining. Preferred methods may be carried out in the context ofdeveloping embryonic tissue, or at an aseptic, unscarred wound site inpost-embryonic tissue. Methods of identifying such morphogens, ormorphogen receptor agonists, are known in the art and include assays forcompounds which induce morphogen-mediated responses (e.g., induction ofendochondral bone formation, induction of differentiation of metanephricmesenchyme, and the like). In a preferred embodiment, morphogens of theinvention, when implanted in a mammal in conjunction with a matrixpermissive of bone morphogenesis, are capable of inducing adevelopmental cascade of cellular and molecular events that culminatesin endochondral bone formation. See, U.S. Pat No. 4,968,590; Sampath, etal., Proc. Natl. Acad. Sci. USA 80: 6591-6595 (1983), the disclosures ofwhich are incorporated by reference herein.

[0031] In an alternative preferred embodiment, morphogens of theinvention are also capable of stimulating production of cell adhesionmolecules, including nerve cell adhesion molecules (N-CAMs). In apreferred embodiment, the present morphogens are capable of stimulatingthe production of N-CAM in vitro in NG108-15 cells, which are apreferred model system for assessing neuronal differentiation,particularly motor neuron differentiation.

[0032] In still other embodiments, an agent which acts as an agonist ofa morphogen receptor may be administered instead of the morphogenitself. An “agonist” of a receptor is a compound which binds to thereceptor, and for which the result of such binding is similar to theresult of binding the natural, endogenous ligand of the receptor. Thatis, the compound must, upon interaction with the receptor, produce thesame or substantially similar transmembrane and/or intracellular effectsas the endogenous ligand. Thus, an agonist of a morphogen receptor bindsto the receptor and such binding has the same or a functionally similarresult as morphogen binding (e.g., induction of morphogenesis). Theactivity or potency of an agonist can be less than that of the naturalligand, in which case the agonist is said to be a “partial agonist,” orit can be equal to or greater than that of the natural ligand, in whichcase it is said to be a “full agonist.” Thus, for example, a smallpeptide or other molecule which can mimic the activity of a morphogen inbinding to and activating the morphogen's receptor may be employed as anequivalent of the morphogen. Preferably the agonist is a full agonist,but partial morphogen receptor agonists may also be advantageouslyemployed. Such an agonist may also be referred to as a morphogen“mimic,” “mimetic,” or “analog.”

[0033] Morphogen inducers and agonists can be identified by mutation,site-specific mutagenesis, combinatorial chemistry, etc. Such methodsare well known in the art. For example, methods of identifying morphogeninducers or agonists of morphogen receptors may be found in U.S. Pat.No. 08/478,097 filed Jun. 7, 1995 and U.S. Pat. No. 08/507,598 filedJul. 26, 1995, the disclosures of which are incorporated herein byreference. Candidate morphogen inducers and agonists are then tested fortheir ability to induce endochondral bone formation and preferably, tostimulate N-CAM production in neurons or in a neuronal model system,such as NG108-15 cells. Morphogen inducers and agonists identifiedaccording to the present invention are capable of inducing endochondralbone formation when implanted in a mammal in conjunction with a matrixpermissive of bone morphogenesis and are capable of stimulatingproduction of N-CAM in vitro.

[0034] The preferred methods, material, and examples that will now bedescribed are illustrative only and are not intended to be limiting.Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 is a tabular presentation of the percent amino acidsequence identity and percent amino acid sequence homology(“similarity”) that various members of the family of morphogenicproteins as defined herein share with hOP-1 in the C-terminal sevencysteine skeleton;

[0036]FIG. 2 (Panels A and B) are photographs illustrating the abilityof morphogen (OP-1) to induce transformed neuroblastoma x glioma cells(Panel 1A) to redifferentiate to a morphology characteristic ofuntransformed neurons (Panel 1B);

[0037]FIG. 3A is a line graph depicting a dose response curve for theinduction of the 180 kDa and 140 kDa N-CAM isoforms in morphogen-treatedNG108-15 cells;

[0038]FIG. 3B is a photograph of a Western blot of whole cell extractsfrom morphogen-treated NG108-15 cells with an N-CAM-specific antibody;and

[0039]FIG. 4 is a line graph depicting the mean number of cellaggregates counted in twenty (20) randomly selected magnified viewingfields as a function of morphogen concentration.

[0040]FIG. 5 is a photograph of an immunoblot demonstrating the presenceof OP-1 in human serum.

[0041]FIG. 6 is a bar graph comparing the effects of OP-1 and glialcells on axonal and dendritic length after one day in vitro.

[0042]FIG. 7 is a bar graph comparing the effects of OP-1 and glialcells on axonal and dendritic length after three days in vitro.

[0043]FIG. 8 is a bar graph comparing the effects of OP-1 and glialcells on dendritic branching after three days in vitro.

[0044]FIG. 9 (Panels A-C) are line graphs depicting a time course of theresponse of cultured sympathetic neurons to OP-1. Intracellular dyeinjections (N>30 for each point) were performed at various times todetermine: the percentage of cells with dendrites (Panel A); the meannumber of dendrites/cell (Panel B); and the number of axons/cell (PanelC). The bars shown in Panel B represent the SEM; where bars are notshown, the SEM was smaller than the size of the symbol. Open symbols,control; filled symbols, cells supplemented with 100 ng/ml OP-1 duringthe time course study.

[0045]FIG. 10 is a line graph depicting the effects of varyingconcentrations of OP-1 on dendritic growth. Sympathetic neurons wereexposed to OP-1 in culture for three days and then immunostained with adendrite-specific mAb (SMI 32). Percentage of cells with dendrites, opencircles; mean number of dendrites per cell, filled circles.

[0046]FIG. 11 is a line graph depicting the effects of varyingconcentrations of different morphogens on dendritic growth. Sympatheticneurons were exposed to various concentrations of BMP-2, OP-1, 60A,BMP-3 or CDMP-2 beginning on the fifth day in vitro and thenimmunostained on day 10 with a dendrite-specific antibody (SMI 32). Dataare presented as the mean±SEM. N=30.

[0047]FIG. 12 is a bar graph comparing the effects of OP-1 and glialcells on synapse formation after three and four days in vitro.

[0048]FIG. 13 is a bar graph depicting the effects of OP-1 treatment onthe size of spinal cord neurons transplanted intra-ocularly in vivo overa period of four weeks.

[0049]FIG. 14 is a photograph of the neurofilament staining ofintra-ocular cultures. Spinal cord transplant cultures were stained fourweeks post-grafting. Cultures were treated with weekly injections ofvehicle or OP-1.

[0050]FIG. 15 is a photograph of the choline acetyltransferase stainingof intra-ocular cultures. Spinal cord transplant cultures were stainedfour weeks post-grafting. Cultures were treated with weekly injectionsof vehicle or OP-1.

[0051]FIG. 16 is a bar graph depicting injury severity scores in theforelimb placing task of sham animals (N=7; black bars), vehicle-treatedanimals with traumatic brain injury (N=8; grey bars), and OP-1-treatedanimals with traumatic brain injury (10 μg/intracistemal injection;total OP-1 delivered in 2 injections=20 μg/animal; N=7; white bars).

[0052]FIG. 17 is a bar graph depicting failure scores in the beam walktask of sham animals (N=5; black bars), vehicle-treated animals withtraumatic brain injury (N=5; grey bars), and OP1-treated animals withtraumatic brain injury (10 μg/intracistemal injection; total OP-1delivered in 2 injections=20 μg/animal; N=4; white bars).

[0053]FIG. 18 is a bar graph depicting beam latency scores of shamanimals (N=5; black bars), vehicle-treated animals with traumatic braininjury (N=5; grey bars), and OP-1-treated animals with traumatic braininjury (10 μg/intracisternal injection; total OP-1 delivered in 2injections=20 μg/animal; N=4; white bars).

DETAILED DESCRIPTION OF THE INVENTION

[0054] It has now been discovered that morphogens enhance survival ofneurons, and maintain neural pathways. As described herein, morphogensare capable of enhancing survival of neurons, stimulating neuronal CAMexpression, maintaining the phenotypic expression of differentiatedneurons, inducing the redifferentiation of transformed cells of neuralorigin, and stimulating axonal growth over breaks in neural processes,particularly large gaps in axons. Morphogens also protect against tissuedestruction associated with immunologically-related nerve tissue damage.Finally, morphogens may be used as part of a method for monitoring theviability of nerve tissue in a mammal.

A. Biochemical, Structural and Functional Properties of UsefulMorphogenic Proteins

[0055] As noted above, a protein is morphogenic as defined herein if itinduces the developmental cascade of cellular and molecular events thatculminate in the formation of new, organ-specific tissue. In a preferredembodiment, a morphogen is a dimeric protein, each polypeptide componentof which has a sequence that corresponds to, or is functionallyequivalent to, at least the conserved C-terminal six or seven cysteineskeleton of human OP-1, included in SEQ ID NO:2, and/or which shares 70%amino acid sequence homology with OP1 in this region. The morphogens aregenerally competent to induce a cascade of events including thefollowing, in a morphogenically permissive environment: stimulatingproliferation of progenitor cells; stimulating the differentiation ofprogenitor cells; stimulating the proliferation of differentiated cells;and supporting the growth and maintenance of differentiated cells. Underappropriate conditions morphogens are also competent to induceredifferentiation of cells that have undergone abnormal differentiation.Details of how the morphogens useful in this invention were identified,as well as a description on how to make, use and test them formorphogenic activity are disclosed in numerous publications, includingU.S. Pat. Nos. 5,011,691 and 5,266,683, and the international patentapplication publications WO 92/15323; WO 93/04692; and WO 94/03200, eachof which are incorporated by reference herein. As disclosed therein, themorphogens can be purified from naturally-sourced material orrecombinantly produced from prokaryotic or eukaryotic host cells, usingthe genetic sequences disclosed therein. Alternatively, novelmorphogenic sequences can be identified following the proceduresdisclosed therein.

[0056] The naturally-occurring morphogens share substantial amino acidsequence homology in their C-terminal sequences (sharing e.g., a six orseven cysteine skeleton sequence). Typically, a naturally-occurringmorphogen is translated as a precursor, having an N-terminal signalpeptide sequence, typically less than about 35 residues in length,followed by a “pro” domain that is cleaved to yield the maturepolypeptide, which includes the biologically active C-terminal skeletonsequence. The signal peptide is cleaved rapidly upon translation, at acleavage site that can be predicted in a given sequence using the methodof Von Heijne, Nucleic Acids Research 14: 4683-4691 (1986). The propolypeptide typically is about three times larger than the fullyprocessed, mature C-terminal polypeptide. Under native conditions, theprotein is secreted as a mature dimer and the cleaved pro polypeptide isthought to remain associated therewith to form a protein complex,presumably to improve the solubility of the mature dimeric protein. Thecomplexed form of a morphogen is generally observed to be more solublethan the mature form under physiological conditions.

[0057] Natural-sourced morphogenic protein in its mature, native form,is typically a glycosylated dimer, having an apparent molecular weightof about 30-36 kDa as determined by SDS-PAGE. When reduced, the 30 kDaprotein gives rise to two glycosylated polypeptide subunits havingapparent molecular weights in the range of about 16 kDa and about 18kDa. The unglycosylated dimeric protein, which also has morphogenicactivity, typically has an apparent molecular weight in the range ofabout 27 kDa. When reduced, the 27 kDa protein gives rise to twounglycosylated polypeptides having molecular weights typically in therange of about 14 kDa to about 16 kDa.

[0058] In preferred embodiments, each of the polypeptide subunits of adimeric morphogenic protein as defined herein comprises an amino acidsequence sharing a defined relationship with an amino acid sequence of areference morphogen. In one embodiment, preferred morphogenicpolypeptide chains share a defined relationship with a sequence presentin morphogenically-active human OP-1, SEQ ID NO:2. However, any one ormore of the naturally-occurring or biosynthetic morphogenic proteinsdisclosed herein similarly could be used as a reference sequence.Preferred morphogenic polypeptide chains share a defined relationshipwith at least the C-terminal six cysteine skeleton of human OP-1,residues 335-431 of SEQ ID NO:2. Preferably, morphogenic proteins sharea defined relationship with at least the C-terminal seven cysteineskeleton of human OP-1, residues 330-431 of SEQ ID NO:2.

[0059] Functionally equivalent sequences include functionally equivalentarrangements of cysteine residues disposed within the referencesequence, including amino acid insertions or deletions which alter thelinear arrangement of these cysteines, but do not materially impairtheir relationship in the folded structure of the dimeric morphogenprotein, including their ability to form such intra- or inter-chaindisulfide bonds as may be necessary for morphogenic activity. Forexample naturally-occurring morphogens have been described in which atleast one internal deletion (of one residue; BMP2) or insertion (of fourresidues; GDF-1) is present but does not abrogate biological activity.Functionally equivalent sequences further include those wherein one ormore amino acid residues differ from the corresponding residue of areference sequence, e.g., the C-terminal seven cysteine skeleton ofhuman OP-1, provided that this difference does not destroy tissuemorphogenic activity. Accordingly, conservative substitutions ofcorresponding amino acids in the reference sequence are preferred. Aminoacid residues that are “conservative substitutions” for correspondingresidues in a reference sequence are those that are physically orfunctionally similar to the corresponding reference residues, e.g., thathave similar size, shape, electric charge, chemical properties includingthe ability to form covalent or hydrogen bonds, or the like.Particularly preferred conservative substitutions are those fulfillingthe criteria defined for an accepted point mutation in Dayhoff, et al.,5 ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, Suppl. 3, ch. 22 pp. 354-352(1978), Natl. Biomed. Res. Found., Washington, D.C. 20007, the teachingsof which are incorporated by reference herein. Examples of conservativesubstitutions include the substitution of one amino acid for anotherwith similar characteristics, e.g., substitutions within the followinggroups: valine, glycine; glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. The term “conservativesubstitution” also includes the use of a synthetic or derivatized aminoacid in place of the corresponding natural parent amino acid, providedthat antibodies raised to the resulting variant polypeptide alsoimmunoreact with the corresponding naturally sourced morphogenpolypeptide.

[0060] The following publications disclose publications morphogenpolypeptide sequences, as well as relevant chemical and physicalproperties, of naturally-occurring and/or synthetic morphogens: OP-1 andOP-2: U.S. Pat. NOS. 5,011,691, 5,266,683, Ozkaynak, et al., EMBO J. 9:2085-2093 (1990); OP-3: WO 94/10203 (PCT US93/10520); BMP-2, BMP-3, andBMP-4: WO 88/00205, Wozney, et al., Science 242: 1528-1534 (1988); BMP-5and BMP-6: Celeste, et al., PNAS 87: 9843-9847 (1991); Vgr-1: Lyons, etal., PNAS 86: 4554-4558 (1989); DPP: Padgett, et al., Nature 325: 81-84(1987); Vg-1: Weeks Cell 51: 861-867 (1987); BMP-9: WO 95/33830(PCT/US95/07084); BMP-10: WO 94/26893 (PCT/US94/05290); BMP-11: WO94/26892 (PCT/US94/05288); BMP-12: WO 95/16035 (PCT/US94/14030); BMP-13:WO 95/16035 (PCT/US94/14030); GDF-1: WO 92/00382 (PCT/US91/04096) andLee, et al., PNAS 88: 4250-4254 (1991); GDF-8: WO 94/21681(PCT/US94/03019); GDF-9: WO 94/15966 (PCT/US94/00685); GDF-10: WO95/10539 (PCT/US94/11440); GDF-11: WO 96/01845 (PCT/US95/08543); BMP-15:WO 96/36710 (PCT/US96/06540); MP121: WO 96/01316 (PCT/EP95/02552); GDF-5(CDMP-1, MP52): WO 94/15949 (PCT/US94/00657) and WO 96/14335(PCT/US94/12814) and WO 93/16099 (PCT/EP93/00350); GDF-6 (CDMP-2,BMP-13): WO 95/01801 (PCT/US94/07762) and WO 96/14335 and WO 95/10635(PCT/US94/14030); GDF-7 (CDMP-3, BMP-12): WO 95/10802 (PCT/US94/07799)and WO 95/10635 (PCT/US94/14030). In another embodiment, useful proteinsinclude biologically active biosynthetic constructs, including novelbiosynthetic morphogenic proteins and chimeric proteins designed usingsequences from two or more known morphogens. See also the biosyntheticconstructs disclosed in U.S. Pat. No. 5,011,691, the disclosure of whichis incorporated herein by reference (e.g., COP-1, COP-3, COP-4, COP-5,COP-7, and COP-16).

[0061] In certain preferred embodiments, useful morphogenic proteinsinclude those in which the amino acid sequences comprise a sequencesharing at least 70% amino acid sequence homology or “similarity”, andpreferably 80% homology or similarity, with a reference morphogenicprotein selected from the exemplary, naturally-occurring morphogenicproteins listed herein. Preferably, the reference protein is human OP-1,and the reference sequence thereof is the C-terminal seven cysteineskeleton present in osteogenically active forms of human OP-1, residues330-431 of SEQ ID NO:2. Useful morphogenic proteins accordingly includeallelic, phylogenetic counterpart and other variants of the preferredreference sequence, whether naturally-occurring or biosyntheticallyproduced (e.g., including “muteins” or “mutant proteins”), as well asnovel members of the general morphogenic family of proteins includingthose set forth and identified above. Certain particularly preferredmorphogenic polypeptides share at least 60% amino acid identity with thepreferred reference sequence of human OP-1, still more preferably atleast 65% amino acid identity therewith.

[0062] In certain embodiments, a polypeptide suspected of beingfunctionally equivalent to a reference morphogen polypeptide is alignedtherewith using the method of Needleman, et al., J. Mol. Biol. 48:443-453 (1970), implemented conveniently by computer programs such asthe Align program (DNAstar, Inc.). As noted above, internal gaps andamino acid insertions in the candidate sequence are ignored for purposesof calculating the defined relationship, conventionally expressed as alevel of amino acid sequence homology, or identity, between thecandidate and reference sequences. “Amino acid sequence homology” isunderstood herein to include both amino acid sequence identity andsimilarity. Homologous sequences share identical and/or similar aminoacid residues, where similar residues are conservation substitutionsfor, or “allowed point mutations” of, corresponding amino acid residuesin an aligned reference sequence. Thus, a candidate polypeptide sequencethat shares 70% amino acid homology with a reference sequence is one inwhich any 70% of the aligned residues are either identical to, or areconservative substitutions of, the corresponding residues in a referencesequence. In a preferred embodiment, the reference sequence is theC-terminal seven cysteine skeleton sequence of human OP-1.

[0063]FIG. 1 recites the percent amino acid sequence homology(similarity) and percent identity within the C-terminal seven cysteineskeletons of various representative members of the TGF-β family, usingOP1 as the reference sequence. The percent homologies recited in thefigure are determined by aligning the sequences essentially followingthe method of Needleman, et al., J. Mol. Biol., 48: 443-453 (1970), andusing the Align Program (DNAstar, Inc.). Insertions and deletions fromthe reference morphogen sequence (the C-terminal, biologically activeseven-cysteine skeleton of hOP-1) are ignored for purposes ofcalculation.

[0064] As is apparent to one of ordinary skill in the art reviewing thesequences for the proteins listed in FIG. 1, significant amino acidchanges can be made from the reference sequence while retainingsubstantial morphogenic activity. For example, while the GDF-1 proteinsequence shares only about 50% amino acid identity with the hOP-1sequence described herein, the GDF-1 sequence shares greater than 70%amino acid sequence homology with the hOP-1 sequence, where “homology”is as defined above. Moreover, GDF-1 contains a four amino acid insert(Gly-Gly-Pro-Pro) between the two residues corresponding to residue 372and 373 of OP-1 (SEQ ID NO:2). Similarly, BMP-3 has a “extra” residue, avaline, inserted between the two residues corresponding to residues 385and 386 of hOP-1 (SEQ ID NO:2). Also, BMP-2 and BMP-4 are both “missing”the amino acid residue corresponding to residue 389 of OP1 (SEQ IDNO:2). None of these “deviations” from the reference sequence appear tointerfere substantially with biological activity.

[0065] In other preferred embodiments, the family of morphogenicpolypeptides useful in the present invention, and members thereof, aredefined by a generic amino acid sequence. For example, Generic Sequence7 (SEQ ID NO:4) and Generic Sequence 8 (SEQ ID NO:5) disclosed below,encompass the observed variations between preferred protein familymembers identified to date, including at least OP-1, OP-2, OP-3,CBMP-2A, CBMP-2B, BMP-3, 60A, DPP, Vg1, BMP-5, BMP-6, Vgr-1, and GDF-1.The amino acid sequences for these proteins are described herein and/orin the art, as summarized above. The generic sequences include both theamino acid identity shared by these sequences in the C-terminalskeleton, defined by the six and seven cysteine skeletons (GenericSequences 7 and 8, respectively), as well as alternative residues forthe variable positions within the sequence. The generic sequencesprovide an appropriate cysteine skeleton where inter- or intramoleculardisulfide bonds can form, and contain certain critical amino acidslikely to influence the tertiary structure of the folded proteins. Inaddition, the generic sequences allow for an additional cysteine atposition 36 (Generic Sequence 7) or position 41 (Generic Sequence 8),thereby encompassing the morphogenically-active sequences of OP-2 andOP-3. Generic Sequence 7 (SEQ ID NO:4)             Leu Xaa Xaa Xaa PheXaa Xaa              1               5 Xaa Gly Trp Xaa Xaa Xaa Xaa XaaXaa Pro         10                  15 Xaa Xaa Xaa Xaa Ala Xaa Tyr CysXaa Gly         20                  25 Xaa Cys Xaa Xaa Pro Xaa Xaa XaaXaa Xaa         30                  35 Xaa Xaa Xaa Asn His Ala Xaa XaaXaa Xaa         40                  45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa         50                  55 Xaa Xaa Xaa Cys Cys Xaa Pro XaaXaa Xaa         60                  65 Xaa Xaa Xaa Xaa Xaa Leu Xaa XaaXaa Xaa         70                  75 Xaa Xaa Xaa Val Xaa Leu Xaa XaaXaa Xaa         80                  85 Xaa Met Xaa Val Xaa Xaa Cys XaaCys Xaa         90                  95

[0066] wherein each Xaa independently is selected from a group of one ormore specified amino acids defined as follows. “Res.” means “residue”and Xaa at res. 2=(Tyr or Lys); Xaa at res. 3=Val or Ilc); Xaa at res.4=(Ser, Asp or Glu); Xaa at res. 6=(Arg, Gln, Ser, Lys or Ala); Xaa atres. 7=(Asp or Glu); Xaa at res. 8=(Leu, Val or Ile); Xaa at res.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res. 12=(Asp, Arg, Asn orGlu); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ile or Val); Xaa atres. 15=(Ile or Val); Xaa at res. 16 (Ala or Ser); Xaa at res. 18=(Glu,Gln, Leu, Lys, Pro or Arg); Xaa at res. 19=(Gly or Ser); Xaa at res.20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Asp, Met, His, Gln, Leu orGly); Xaa at res. 23=(Tyr, Asn or Phe); Xaa at res. 26=(Glu, His, Tyr,Asp, Gln, Ala or Ser); Xaa at res. 28=(Glu, Lys, Asp, Gln or Ala); Xaaat res. 30=(Ala, Ser, Pro, Gln, Ile or Asn) Xaa at res. 31=(Phe, Leu orTyr); Xaa at res. 33=(Leu, Val or Met); Xaa at res. 34=(Asn, Asp, Ala,Thr or Pro); Xaa at res. 35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa atres. 36=(Tyr, Cys, His, Ser or Ile); Xaa at res. 37=(Met, Phe, Gly orLeu); Xaa at res. 38=(Asn, Ser or Lys); Xaa at res. 39=(Ala, Ser, Gly orPro); Xaa at res. 40=(Thr, Leu or Ser); Xaa at res. 44=(Ile, Val orThr); Xaa at res. 45=(Val, Leu, Met or Ile); Xaa at res. 46=(Gln orArg); Xaa at res. 47=(Thr, Ala or Ser); Xaa at res. 48=(Leu or Ile); Xaaat res. 49=(Val or Met); Xaa at res. 50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res. 52=(Ile, Met, Asn, Ala,Val, Gly or Leu); Xaa at res. 53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaaat res. 54=(Pro, Ser or Val); Xaa at res. 55=(Glu, Asp, Asn, Gly, Val,Pro or Lys); Xaa at res. 56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ileor His); Xaa at res. 57=(Val, Ala or Ile); Xaa at res. 58=(Pro or Asp);Xaa at res. 59=(Lys, Leu or Glu); Xaa at res. 60=(Pro, Val or Ala); Xaaat res. 63=(Ala or Val); Xaa at res. 65=(Thr, Ala or Glu); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res. 67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser, Asp or Gly); Xaa at res. 69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res. 71=(Ser, Ala or Pro); Xaa at res.72=(Val, Leu, Met or Ile); Xaa at res. 74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res. 76=(Asp, Asn or Leu); Xaa at res.77=(Asp, Glu, Asn, Arg or Ser); Xaa at res. 78=(Ser, Gln, Asn, Tyr orAsp); Xaa at res. 79=(Ser, Asn, Asp, Glu or Lys); Xaa at res. 80=(Asn,Thr or Lys); Xaa at res. 82=(Ile, Val or Asn); Xaa at res. 84=(Lys orArg); Xaa at res. 85=(Lys, Asn, Gln, His, Arg or Val); Xaa at res.86=(Tyr, Glu or His); Xaa at res. 87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu, Trp or Asp); Xaa at res. 90=(Val, Thr, Ala or Ile); Xaa atres. 92=(Arg, Lys, Val, Asp, Gln or Glu); Xaa at res. 93=(Ala, Gly, Gluor Ser); Xaa at res. 95=(Gly or Ala) and Xaa at res. 97=(His or Arg).

[0067] Generic Sequence 8 (SEQ ID NO:5) includes all of Generic Sequence7 (SEQ ID NO:4) and in addition includes the following sequence (SEQ IDNO:8) at its N-terminus: SEQ ID NO: 8 Cys Xaa Xaa Xaa Xaa 1               5

[0068] Accordingly, beginning with residue 7, each “Xaa” in GenericSequence 8 is a specified amino acid defined as for Generic Sequence 7,with the distinction that each residue number described for GenericSequence 7 is shifted by five in Generic Sequence 8. Thus, “Xaa at res.2=(Tyr or Lys)” in Generic Sequence 7 refers to Xaa at res. 7 in GenericSequence 8. In Generic Sequence 8, Xaa at res. 2=(Lys, Arg, Ala or Gln);Xaa at res. 3=(Lys, Arg or Met); Xaa at res. 4=(His, Arg or Gln); andXaa at res. 5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).

[0069] In another embodiment, useful osteogenic proteins include thosedefined by Generic Sequences 9 and 10 (SEQ ID NOS:6 and 7,respectively), described herein above. Specifically, Generic Sequences 9and 10 are composite amino acid sequences of the following proteins:human OP-1, human OP-2, human OP-3, human BMP-2, human BMP-3, humanBMP-4, human BMP-5, human BMP-6, human BMP-8, human BMP-9, human BMP-10,human BMP-11, Drosophila 60A, Xenopus Vg-1, sea urchin UNIVIN, humanCDMP-1 (mouse GDF-5), human CDMP-2 (mouse GDF-6, human BMP-13), humanCDMP-3 (mouse GDF-7, human BMP-12), mouse GDF-3, human GDF-1, mouseGDF-1, chicken DORSALIN, Drosophila dpp, Drosophila SCREW, mouse NODAL,mouse GDF-8, human GDF-8, mouse GDF-9, mouse GDF-10, human GDF-11, mouseGDF-11, human BMP-15, and rat BMP-3b. Like Generic Sequence 7, GenericSequence 9 accommodates the C-terminal six cysteine skeleton and, likeGeneric Sequence 8, Generic Sequence 10 accommodates the seven cysteineskeleton. Generic Sequence 9 (SEQ ID NO:6) Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa  1               5                  10 Xaa Xaa Xaa Xaa XaaXaa Pro Xaa Xaa Xaa                 15                  20 Xaa Xaa XaaXaa Cys Xaa Gly Xaa Cys Xaa                 25                  30 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                35                  40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa                 45                  50 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa                 55                  60 Xaa Cys Xaa ProXaa Xaa Xaa Xaa Xaa Xaa                 65                  70 Xaa XaaLeu Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 75                  80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                85                  90 Xaa Xaa Xaa Cys Xaa Cys Xaa                95

[0070] wherein each Xaa is independently selected from a group of one ormore specified amino acids defined as follows: “Res.” means “reside” andXaa at res. 1=(Phe, Leu or Glu); Xaa at res. 2=(Tyr, Phe, His, Arg, Thr,Lys, Gln, Val or Glu); Xaa at res. 3=(Val, Ile, Leu or Asp); Xaa at res.4=(Ser, Asp, Glu, Asn or Phe); Xaa at res. 5=(Phe or Glu); Xaa at res.6=(Arg, Gln, Lys, Ser, Glu, Ala or Asn); Xaa at res. 7=(Asp, Glu, Leu,Ala or Gln); Xaa at res. 8=(Leu, Val, Met, Ile or Phe); Xaa at res.9=(Gly, His or Lys); Xaa at res. 10=(Trp or Met); Xaa at res. 11=(Gln,Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at res. 12=(Asp, Asn, Ser,Lys, Arg, Cgu or His); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ileor Val); Xaa at res. 15=(Ile or Val); Xaa at res. 16=(Ala, Ser, Tyr orTrp); Xaa at res. 18=(Glu, Lys, Gln, Met, Pro, Leu, Arg, His or Lys);Xaa at res. 19=(Gly, Glu, Asp, Lys, Ser, Gln, Arg or Phe); Xaa at res.20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Gly, Met, Gln, His, Glu, AspLeu, Asn, Lys or Thr); Xaa at res. 22=(Ala or Pro); Xaa at res. 23=(Tyr,Phe, Asn, Ala or Arg); Xaa at res. 24=(Tyr, His, Glu, Phe or Arg); Xaaat res. 26=(Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gln or Gly); Xaa atres. 28=(Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gln); Xaa at res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp, Phe, Gln or Leu); Xaa at res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at res. 32=(Pro, Ser, Ala orVal); Xaa at res. 33=(Leu, Met, Glu, Phe or Val); Xaa at res. 34=(Asn,Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at res. 35=(Ser, Ala, Glu,Asp, Thr, Leu, Lys, Gln or His); Xaa at res. 36=(Tyr, His, Cys, Ile,Arg, Asp, Asn, Lys, Ser, Glu or Gly); Xaa at res. 37=(Met, Leu, Phe,Val, Gly or Tyr); Xaa at res. 38=(Asn, Glu, Thr, Pro, Lys, His, Gly,Met, Val or Arg); Xaa at res. 39=(Ala, Ser, Gly, Pro or Phe); Xaa atres. 40=(Thr, Ser, Leu, Pro, His or Met); Xaa at res. 41=(Asn, Lys, Val,Thr or Gln); Xaa at res. 42=(His, Tyr or Lys); Xaa at res. 43=(Ala, Thr,Leu or Tyr); Xaa at res. 44=(Ile, Thr, Val, Phe, Tyr, Met or Pro); Xaaat res. 45=(Val, Leu, Met, Ile or His); Xaa at res. 46=(Gln, Arg orThr); Xaa at res. 47=(Thr, Ser, Ala, Asn or His); Xaa at res. 48=(Leu,Asn or Ile); Xaa at res. 49=(Val, Met, Leu, Pro or Ile); Xaa at res.50=(His, Asn, Arg, Lys, Tyr or Gln); Xaa at res. 51=(Phe, Leu, Ser, Asn,Met, Ala, Arg, Glu, Gly or Gln); Xaa at res. 52=(Ile, Met, Leu, Val,Lys, Gln, Ala or Tyr); Xaa at res. 53=(Asn, Phe, Lys, Glu, Asp, Ala,Gln, Gly, Leu or Val); Xaa at res. 54=(Pro, Asn, Ser, Val or Asp); Xaaat res. 55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gln, Pro or His);Xaa at res. 56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaaat res. 57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res. 58=(Pro, Gly,Ser, Asp or Ala); Xaa at res. 59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg orGly); Xaa at res. 60=(Pro, Ala, Val, Thr or Ser); Xaa at res. 61=(Cys,Val or Ser); Xaa at res. 63=(Ala, Val or Thr); Xaa at res. 65=(Thr, Ala,Glu, Val, Gly, Asp or Tyr); Xaa at res. 66=(Gln, Lys, Glu, Arg or Val);Xaa at res. 67=(Leu, Met, Thr or Tyr); Xaa at res. 68=(Asn, Ser, Gly,Thr, Asp, Glu, Lys or Val); Xaa at res. 69=(Ala, Pro, Gly or Ser); Xaaat res. 70=(Ile, Thr, Leu or Val); Xaa at res. 71=(Ser, Pro, Ala, Thr,Asn or Gly); Xaa at res. 2=(Val, Ile, Leu or Met); Xaa at res. 74=(Tyr,Phe, Arg, Thr, Tyr or Met); Xaa at res. 75=(Phe, Tyr, His, Leu, Ile,Lys, Gln or Val); Xaa at res. 76=(Asp, Leu, Asn or Glu); Xaa at res.77=(Asp, Ser, Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res. 78=(Ser,Asn, Asp, Tyr, Ala, Gly, Gln, Met, Glu, Asn or Lys); Xaa at res.79=(Ser, Asn, Glu, Asp, Val, Lys, Gly, Gln or Arg); Xaa at res. 80=(Asn,Lys, Thr, Pro, Val, Ile, Arg, Ser or Gln); Xaa at res. 81=(Val, Ile, Thror Ala); Xaa at res. 82=(Ile, Asn, Val, Leu, Tyr, Asp or Ala); Xaa atres. 83=(Leu, Tyr, Lys or Ile); Xaa at res. 84=(Lys, Arg, Asn, Tyr, Phe,Thr, Glu or Gly); Xaa at res. 85=(Lys, Arg, His, Gln, Asn, Glu or Val);Xaa at res. 86=(Tyr, His, Glu or Ile); Xaa at res. 87=(Arg, Glu, Gln,Pro or Lys); Xaa at res. 88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa atres. 89=(Met or Ala); Xaa at res. 90=(Val, Ile, Ala, Thr, Ser or Lys);Xaa at res. 91=(Val or Ala); Xaa at res. 92=(Arg, Lys, Gln, Asp, Glu,Val, Ala, Ser or Thr); Xaa at res. 93=(Ala, Ser, Glu, Gly, Arg or Thr);Xaa at res. 95=(Gly, Ala or Thr); Xaa at res. 97=(His, Arg, Gly, Leu orSer). Further, after res. 53 in rBMP-3b and mGDF-10 there is an Ile;after res. 54 in GDF-1 there is a T; after res. 54 in BMP-3 there is aV; after res. 78 in BMP-8 and Dorsalin there is a G; after res. 37 inhGDF-1 there is Pro, Gly, Gly, Pro.

[0071] Generic Sequence 10 (SEQ ID NO:7) includes all of GenericSequence 9 (SEQ ID NO:6) and in addition includes the following sequence(SEQ ID NO:9) at its N-terminus: SEQ ID NO:9 Cys Xaa Xaa Xaa Xaa 1               5

[0072] Accordingly, beginning with residue 6, each “Xaa” in GenericSequence 10 is a specified amino acid defined as for Generic Sequence 9,with the distinction that each residue number described for GenericSequence 9 is shifted by five in Generic Sequence 10. Thus, “Xaa at res.1=( Tyr, Phe, His, Arg, Thr, Lys, Gln, Val or Glu)” in Generic Sequence9 refers to Xaa at res. 6 in Generic Sequence 10. In Generic Sequence10, Xaa at res. 2=(Lys, Arg, Gln, Ser, His, Glu, Ala, or Cys); Xaa atres. 3=(Lys, Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at res. 4=(His,Gln, Arg, Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at res. 5=(Gln, Thr,His, Arg, Pro, Ser, Ala, Gln, Asn, Tyr, Lys, Asp, or Leu).

[0073] Based upon alignment of the naturally-occurring morphogens withinthe definition of Generic Sequence 10, it should be clear that gapsand/or insertions of one or more amino acid residues can be tolerated(without abrogating or substantially impairing biological activity) atleast between or involving residues 11-12, 42-43, 59-60, 68-69 and83-84.

[0074] As noted above, certain preferred morphogenic polypeptidesequences useful in this invention have greater than 60% identity,preferably greater than 65% identity, with the amino acid sequencedefining the preferred reference sequence of hOP-1. These particularlypreferred sequences include allelic and phylogenetic counterpartvariants of the OP-1 and OP-2 proteins, including the Drosophila 60Aprotein, as well as the closely related proteins BMP-5, BMP-6 and Vgr-1.Accordingly, in certain particularly preferred embodiments, usefulmorphogenic proteins include active proteins comprising pairs ofpolypeptide chains within the generic amino acid sequence hereinreferred to as “OPX” (SEQ ID NO:3), which defines the seven cysteineskeleton and accommodates the homologies between several identifiedvariants of OP-1 and OP-2. Accordingly, each “Xaa” at a given positionin OPX independently is selected from the residues occurring at thecorresponding position in the C-terminal sequence of mouse or human OP-1or OP-2. Specifically, each “Xaa” is independently selected from a groupof one or more specified amino acids as defined below: Cys Xaa Xaa HisGlu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp 1               5                   10                  15 Xaa Asp TrpXaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys                 20                  25                  30 Glu Gly GluCys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr                 35                  40                  45 Asn His AlaIle Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa                 50                  55                  60 Xaa Val ProLys Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa                 65                  70                  75 Ser Val LeuTyr Xaa Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys                 80                  85                  90 Xaa Arg AsnMet Val Val Xaa Ala Cys Gly Cys His                 95                 100

[0075] wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys or Arg);Xaa at res. 11=(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa at res.19=(Ile or Val); Xaa at res. 23=(Glu or Gln); Xaa at res. 26=(Ala orSer); Xaa at res. 35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa atres. 41=(Tyr or Cys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Seror Thr); Xaa at res. 56=(Phe or Leu); Xaa at res. 57=(Ile or Met); Xaaat res. 58=(Asn or Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val); Xaa at res. 65=(Pro or Ala); Xaa at res. 71=(Glnor Lys); Xaa at res. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaaat res. 80=(Phe or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr orHis); and Xaa at res. 97=(Arg or Lys).

[0076] In still another preferred embodiment, usefulmorphogenically-active proteins have polypeptide chains with amino acidsequences comprising a sequence encoded by a nucleic acid thathybridizes with DNA or RNA encoding reference morphogen sequences, e.g.,C-terminal sequences defining the conserved seven cysteine skeletons ofOP-1, OP-2, BMP-2, BMP-4, BMP-5, BMP-6, 60A, GDF-3, GDF-5, GDF-6, GDF-7and the like. As used herein, high stringency hybridization conditionsare defined as hybridization according to known techniques in 40%formamide, 5×SSPE, 5×Denhardt's Solution, and 0.1% SDS at 37° C.overnight, and washing in 0.1×SSPE, 0.1% SDS at 50° C. Standardstringency conditions are well characterized in standard molecularbiology cloning texts. See, for example, MOLECULAR CLONING A LABORATORYMANUAL, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold SpringHarbor Laboratory Press: 1989); DNA CLONING, Volumes I and II (D. N.Glover ed., 1985); OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed., 1984);NUCLEIC ACID HYBRIDIZATION (B. D. Hames & S. J. Higgins eds. 1984); andB. Perbal, A PRACTICAL GUIDE To MOLECULAR CLONING (1984).

[0077] In other embodiments, as an alternative to the administration ofa morphogenic protein, an effective amount of an agent competent tostimulate or induce increased endogenous morphogen expression in amammal may be administered by any of the routes described herein. Such amorphogen inducer may be provided to a mammal, e.g., by systemicadministration to the mammal or by direct administration to the neuraltissue. A method for identifying and testing inducers (stimulatingagents) competent to modulate the levels of endogenous morphogens in agiven tissue is described in published applications WO93/05172 andWO93/05751, each of which is incorporated by reference herein. Briefly,candidate compounds are identified and tested by incubation in vitrowith test tissue or cells, or a cultured cell line derived therefrom,for a time sufficient to allow the compound to affect the production, i.e., cause the expression and/or secretion, of a morphogen produced bythe cells of that tissue. Suitable tissue, or cultured cells of asuitable tissue, are preferably selected from renal epithelium, ovariantissue, fibroblasts, and osteoblasts.

[0078] In yet other embodiments, an agent which acts as an agonist of amorphogen receptor may be administered instead of the morphogen itself.Such an agent may also be referred to an a morphogen “mimic,” “mimetic,”or “analog.” Thus, for example, a small peptide or other molecule whichcan mimic the activity of a morphogen in binding to and activating themorphogen's receptor may be employed as an equivalent of the morphogen.Preferably the agonist is a full agonist, but partial morphogen receptoragonists may also be advantageously employed. Methods of identifyingsuch agonists are known in the art and include assays for compoundswhich induce morphogen-mediated responses (e.g., induction ofdifferentiation of metanephric mesenchyme, induction of endochondralbone formation). For example, methods of identifying morphogen inducersor agonists of morphogen receptors may be found in U.S. Pat. No.08/478,097 filed Jun. 7, 1995 and U.S. Pat. No. 08/507,598 filed Jul.26, 1995, disclosures of which are incorporated herein by reference.

[0079] As a general matter, methods of the present invention may beapplied to the treatment of any mammalian subject at risk of orafflicted with a neural tissue insult or neuropathy. The invention issuitable for the treatment of any primate, preferably a higher primatesuch as a human. In addition, however, the invention may be employed inthe treatment of domesticated mammals which are maintained as humancompanions (e.g., dogs, cats, horses), which have significant commercialvalue (e.g., goats, pigs, sheep, cattle, sporting or draft animals),which have significant scientific value (e.g., captive or free specimensof endangered species, or inbred or engineered animal strains), or whichotherwise have value.

C. Formulations and Methods of Treatment

[0080] Morphogens, morphogen inducers, or agonists of morphogenreceptors of the present invention may be administered by any routewhich is compatible with the particular morphogen, inducer, or agonistemployed. Thus, as appropriate, administration may be oral orparenteral, including intravenous and intraperitoneal routes ofadministration. In addition, administration may be by periodicinjections of a bolus of the morphogen, inducer or agonist, or may bemade more continuous by intravenous or intraperitoneal administrationfrom a reservoir which is external (e.g., an i.v. bag) or internal(e.g., a bioerodable implant, or a colony of implanted,morphogen-producing cells).

[0081] Therapeutic agents of the invention (i.e., morphogens, morphogeninducers or agonists of morphogen receptors) may be provided to anindividual by any suitable means, directly (e.g., locally, as byinjection, implantation or topical administration to a tissue locus) orsystemically (e.g., parenterally or orally). Where the agent is to beprovided parenterally, such as by intravenous, subcutaneous,intramolecular, ophthalmic, intraperitoneal, intramuscular, buccal,rectal, vaginal, intraorbital, intracerebral, intracranial, intraspinal,intraventricular, intrathecal, intracisternal, intracapsular, intranasalor by aerosol administration, the agent preferably comprises part of anaqueous or physiologically compatible fluid suspension or solution.Thus, the morphogen carrier or vehicle is physiologically acceptable sothat in addition to delivery of the desired agent to the patient, itdoes not otherwise adversely affect the patient's electrolyte and/orvolume balance. The fluid medium for the agent thus can comprise normalphysiologic saline (e.g., 9.85% aqueous NaCl, 0.15M, pH 7-7.4).

[0082] Association of the mature morphogen dimer with a morphogen prodomain results in the pro form of the morphogen which typically is moresoluble in physiological solutions than the corresponding mature form.In fact, endogenous morphogens are thought to be transported (e.g.,secreted and circulated) in the mammalian body in this form. Thissoluble form of the protein can be obtained from culture medium ofmorphogen-secreting mammalian cells, e.g., cells transfected withnucleic acid encoding and competent to express the morphogen.Alternatively, a soluble species can be formulated by complexing themature, morphogenically-active polypeptide dimer (or an active fragmentthereof) with a morphogen pro domain polypeptide or asolubility-enhancing fragment thereof. Solubility-enhancing pro domainfragments can be any N-terminal, C-terminal or internal fragment of thepro region of a member of the morphogen family that complexes with themature polypeptide dimer to enhance stability and/or dissolubility ofthe resulting noncovalent or convalent complex. Typically, usefulfragments are those cleaved at the proteolytic site Arg-Xaa-Xaa-Arg. Adetailed description of soluble complex forms of morphogenic proteins,including how to make, test and use them, is described in WO 94/03600(PCT US 93/07189). In the case of OP-1, useful pro domain polypeptidefragments include the intact pro domain polypeptide (residues 30-292)and fragments 48-292 and 158-292, all of SEQ ID NO:2. Another moleculecapable of enhancing solubility and particularly useful for oraladministrations, is casein. For example, addition of 0.2% caseinincreases solubility of the mature active form of OP1 by 80%. Othercomponents found in milk and/or various serum proteins may also beuseful.

[0083] Useful solutions for parenteral administration may be prepared byany of the methods well known in the pharmaceutical art, described, forexample, in REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro, A., ed.), MackPub., 1990. Formulations of the therapeutic agents of the invention mayinclude, for example, polyalkylene glycols such as polyethylene glycol,oils of vegetable origin, hydrogenated naphthalenes, and the like.Formulations for direct administration, in particular, may includeglycerol and other compositions of high viscosity to help maintain theagent at the desired locus. Biocompatible, preferably bioresorbable,polymers, including, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, lactide, and glycolide polymers andlactide/glycolide copolymers, may be useful excipients to control therelease of the agent in vivo. Other potentially useful parenteraldelivery systems for these agents include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation administration contain asexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or oily solutions for administration in the form ofnasal drops, or as a gel to be applied intranasally. Formulations forparenteral administration may also include glycocholate for buccaladministration, methoxysalicylate for rectal administration, or cutricacid for vaginal administration. Suppositories for rectal administrationmay also be prepared by mixing the morphogen, inducer or agonist with anon-irritating excipient such as cocoa butter or other compositionswhich are solid at room temperature and liquid at body temperatures.

[0084] Formulations for topical administration to the skin surface maybe prepared by dispersing the morphogen, inducer or agonist with adermatologically acceptable carrier such as a lotion, cream, ointment orsoap. Particularly useful are carriers capable of forming a film orlayer over the skin to localize application and inhibit removal. Fortopical administration to internal tissue surfaces, the agent may bedispersed in a liquid tissue adhesive or other substance known toenhance adsorption to a tissue surface. For example,hydroxypropylcellulose or fibrinogen/thrombin solutions may be used toadvantage. Alternatively, tissue-coating solutions, such aspectin-containing formulations may be used.

[0085] Alternatively, the agents described herein may be administeredorally. Oral administration of proteins as therapeutics generally is notpracticed, as most proteins are readily degraded by digestive enzymesand acids in the mammalian digestive system before they can be absorbedinto the bloodstream. However, the morphogens described herein typicallyare acid stable and protease-resistant (see, for example, U.S. Pat. No.4,968,590). In addition, at least one morphogen, OP-1, has beenidentified in mammary gland extract, colostrum and 57-day milk.Moreover, the OP-1 purified from mammary gland extract ismorphogenically-active and is also detected in the bloodstream. Maternaladministration, via ingested milk, may be a natural delivery route ofTGF-β superfamily proteins. Letterio, et al., Science 264: 1936-1938(1994), report that TGF-β is present in murine milk, and thatradiolabelled TGF-β is absorbed by gastrointestinal mucosa of sucklingjuveniles. Labeled, ingested TGF-β appears rapidly in intact form in thejuveniles' body tissues, including lung, heart and liver. Finally,soluble form morphogen, e.g., mature morphogen associated with the prodomain, is morphogenically-active. These findings, as well as thosedisclosed in the examples below, indicate that oral and parenteraladministration are viable means for administering TGF-β superfamilyproteins, including the morphogens, to an individual. In addition, whilethe mature forms of certain morphogens described herein typically aresparingly soluble, the morphogen form found in milk (and mammary glandextract and colostrum) is readily soluble, probably by association ofthe mature, morphogenically-active form with part or all of the prodomain of the expressed, full length polypeptide sequence and/or byassociation with one or more milk components. Accordingly, the compoundsprovided herein may also be associated with molecules capable ofenhancing their solubility in vitro or in vivo.

[0086] Where the morphogen is intended for use as a therapeutic fordisorders of the CNS, an additional problem must be addressed:overcoming the blood-brain barrier, the brain capillary wall structurethat effectively screens out all but selected categories of substancespresent in the blood, preventing their passage into the brain. Theblood-brain barrier can be bypassed effectively by direct infusion ofthe morphogen or morphogen-stimulating agent into the brain, or byintranasal administration or inhalation of formulations suitable foruptake and retrograde transport by olfactory neurons. Alternatively, themorphogen or morphogen-stimulating agent can be modified to enhance itstransport across the blood-brain barrier. For example, truncated formsof the morphogen or a morphogen-stimulating agent may be mostsuccessful. Alternatively, the morphogens, inducers or agonists providedherein can be derivatized or conjugated to a lipophilic moiety or to asubstance that is actively transported across the blood-brain barrier,using standard means known to those skilled in the art. See, forexample, Pardridge, Endocrine Reviews 7: 314-330 (1986) and U.S. Pat.No. 4,801,575.

[0087] The compounds provided herein may also be associated withmolecules capable of targeting the morphogen, inducer or agonist to thedesired tissue. For example, an antibody, antibody fragment, or otherbinding protein that interacts specifically with a surface molecule oncells of the desired tissue, may be used. Useful targeting molecules maybe designed, for example, using the single chain binding site technologydisclosed in U.S. Pat. No. 5,091,513. Targeting molecules can becovalently or non-covalently associated with the morphogen, inducer oragonist.

[0088] As will be appreciated by one of ordinary skill in the art, theformulated compositions contain therapeutically-effective amounts of themorphogen, morphogen inducers or agonists of morphogen receptors. Thatis, they contain an amount which provides appropriate concentrations ofthe agent to the affected nervous system tissue for a time sufficient tostimulate a detectable restoration of impaired central or peripheralnervous system function, up to and including a complete restorationthereof. As will be appreciated by those skilled in the art, theseconcentrations will vary depending upon a number of factors, includingthe biological efficacy of the selected agent, the chemicalcharacteristics (e.g., hydrophobicity) of the specific agent, theformulation thereof, including a mixture with one or more excipients,the administration route, and the treatment envisioned, includingwhether the active ingredient will be administered directly into atissue site, or whether it will be administered systemically. Thepreferred dosage to be administered is also likely to depend onvariables such as the condition of the diseased or damaged tissues, andthe overall health status of the particular mammal. As a general matter,single, daily, biweekly or weekly dosages of 0.00001-1000 mg of amorphogen are sufficient, with 0.0001-100 mg being preferable, and 0.001to 10 mg being even more preferable. Alternatively, a single, daily,biweekly or weekly dosage of 0.01-1000 μg/kg body weight, morepreferably 0.01-10 mg/kg body weight, may be advantageously employed.The present effective dose can be administered in a single dose or in aplurality (two or more) of installment doses, as desired or consideredappropriate under the specific circumstances. A bolus injection ordiffusable infusion formulation can be used. If desired to facilitaterepeated or frequent infusions, implantation of a semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular)may be advisable.

[0089] The morphogens, inducers or agonists of the invention may, ofcourse, be administered alone or in combination with other moleculesknown to be beneficial in the treatment of the conditions describedherein. For example, various well-known growth factors, hormones,enzymes, therapeutic compositions, antibiotics, or other bioactiveagents can also be administered with the morphogen. Thus, various knowngrowth factors such as NGF, EGF, PDGF, IGF, FGF, TGF-α, and TGF-β, aswell as enzymes, enzyme inhibitors, antioxidants, anti-inflammatoryagents, free radical scavenging agents, antibiotics and/orchemoattractant/chemotactic factors, can be included in the presentmorphogen formulation.

EXAMPLE 1 Preparation of Soluble Morphogen Protein Solutions for In vivoAdministration A. Aqueous Solutions

[0090] While the mature dimeric morphogenic proteins defined herein aretypically sparingly soluble in physiological buffers, they can besolubilized to form injectable suspensions or solutions. One exemplaryaqueous formulation containing a morphogen is made, for example, bydispersing the morphogen in 50% ethanol containing acetonitrile in 0.1%trifluoroacetic acid (TFA) or 0.1% HCl, or in an equivalent solvent. Onevolume of the resultant solution then is added, for example, to tenvolumes of phosphate buffered saline (PBS), which further may include0.1-0.2% human serum albumin (HSA) or a similar carrier protein. Theresultant solution is preferably vortexed extensively to produce aphysiologically acceptable morphogen formulation.

[0091] In another embodiment, the morphogen, including OP-1, issolubilized by reducing the pH of the solution. In one preferredformulation, the protein is solubilized in 0.2 mM acetate buffer, pH4.5, containing 5% mannitol, to render the solution more isotonic. Otherstandard means for creating physiologically acceptable formulations arewithin the skill of the art.

B. Soluble Complex Formulations

[0092] Another preferred form is a dimeric morphogenic proteincomprising at least the C-terminal seven cysteine skeletoncharacteristic of the morphogen family, complexed with a peptidecomprising a pro region of a member of the morphogen family, or asolubility-enhancing fragment thereof, or an allelic, phylogenetic orother sequence variant thereof. The solubility-enhancing fragment is anyN-terminal or C-terminal fragment of the pro domain polypeptide of amember of the morphogen family that complexes with the maturepolypeptide dimer to enhance the stability of the resulting solublecomplex. Preferably, the dimeric morphogenic protein is complexed withtwo such pro domain peptides.

[0093] As described above and in published application WO 94/03600,incorporated by reference herein, the soluble complex form is isolatedfrom the cell culture media (or a body fluid) under appropriateconditions. Alternatively, the complex is formulated in vitro.

[0094] Soluble morphogen complexes are isolated from conditioned mediausing a simple, three step chromatographic protocol performed in theabsence of denaturants. The protocol involves running the media (or bodyfluid) over an affinity column, followed by ion exchange and gelfiltration chromatographies generally as described in WO 94/03600. Theaffinity column described below is a Zn-IMAC column. The present exampleuses human OP-1, and is not intended to be limiting. The presentprotocol has general applicability to the purification of a variety ofmorphogens, all of which are anticipated to be isolatable using onlyminor modifications of the protocol described below. An alternativeprotocol also envisioned to have utility includes an immunoaffinitycolumn, created using standard procedures and, for example, usingantibody specific for a given morphogen pro domain (complexed, forexample, to a protein A-conjugated Sepharose column). Protocols fordeveloping immunoaffinity columns are well described in the art (see,for example, GUIDE TO PROTEIN PURIFICATION, M. Deutscher, ed., AcademicPress, San Diego, 1990, particularly sections VII and XI thereof).

[0095] In this example, OP-1 was expressed in mammalian (CHO, Chinesehamster ovary) cells as described in the art (see, for example,international application US90/05903 (WO 91/05802). The CHO cellconditioned media containing 0.5% FBS is initially purified usingImmobilized Metal-Ion Affinity Chromatography (IMAC). The soluble OP-1complex from conditioned media binds very selectively to the Zn-IMACresin and a high concentration of imidazole (50 mM imidazole, pH 8.0) isrequired for the effective elution of the bound complex. The Zn-IMACpurified soluble OP-1 is next applied to an S-Sepharose action-exchangecolumn equilibrated in 20 mM NaPO₄ (pH 7.0) with 50 mM NaCl. The proteinthen is applied to a Sephacryl S-200HR column equilibrated in TBS. Usingsubstantially the same protocol, soluble morphogens can also be isolatedfrom one or more body fluids, including milk, serum, cerebrospinal fluidor peritoneal fluid.

[0096] The soluble OP-1 complex elutes with an apparent molecular weightof 110 kDa. This agrees well with the predicted composition of thesoluble OP1 complex, with one mature OP1 dimer (35-36 kDa) associatedwith two pro domains (39 kDa each). Purity of the final complex can beverified by running the appropriate fraction in a reduced 15%polyacrylamide gel.

[0097] As an alternative to purifying soluble complexes from culturemedia or a body fluid, soluble complexes can be formulated from purifiedpro domains and mature dimeric species. Successful complex formationapparently requires association of the components under denaturingconditions sufficient to relax the folded structure of these molecules,without affecting disulfide bonds. Preferably, the denaturing conditionsmimic the environment of an intracellular vesicle sufficiently such thatthe cleaved pro domain polypeptide has an opportunity to associate withthe mature dimeric protein under relaxed folding conditions. Theconcentration of denaturant in the solution then is decreased in acontrolled, preferably step-wise manner, so as to allow proper refoldingof the dimer and pro domain peptides, while maintaining the associationof the pro domain peptides with the mature dimer. Useful denaturantsinclude 4-6 M urea or guanidine hydrochloride (GuHCl), in bufferedsolutions of pH 4-10, preferably pH 6-8. The soluble complex then isformed by controlled dialysis or dilution into a solution having a finaldenaturant concentration of less than 0.1 -2M urea or GuHCl, preferably1-2 M urea or GuHCl, which then preferably can be diluted into aphysiological buffer. Protein purification/renaturing procedures andconsiderations are well described in the art, and details for developinga suitable renaturing protocol readily can be determined by one havingordinary skill in the art. One useful text on the subject is GUIDE TOPROTEIN PURIFICATION, M. Deutscher, ed., Academic Press, San Diego,1990, particularly section V. Complex formation may also be aided byaddition of one or more chaperone proteins.

[0098] The stability of the highly purified soluble morphogen complex ina physiological buffer, e.g., Tris-buffered saline (TBS) andphosphate-buffered saline (PBS), can be enhanced by any of a number ofmeans, including any one or more of three classes of additives. Theseadditives include basic amino acids (e.g., L-arginine, lysine andbetaine); nonionic detergents (e.g., Tween 80 or NonIdet P-120); andcarrier proteins (e.g., serum albumin and casein). Useful concentrationsof these additives include 1-100 mM, preferably 10-70 mM, including 50mM, basic amino acid; 0.01-1.0%, preferably 0.05-0.2%, including 0.1%(v/v) nonionic detergent; and 0.01-1.0%, preferably 0.05-0.2%, including0.1% (w/v) carrier protein.

EXAMPLE 2 Identification of Morphogen-expressing Tissue

[0099] Determining the tissue distribution of morphogens may be used toidentify different morphogens expressed in a given tissue, as well as toidentify new, related morphogens. Tissue distribution also may be usedto identify useful morphogen-producing tissue for use in screening andidentifying candidate morphogen-stimulating agents. The morphogens (ortheir mRNA transcripts) readily are identified in different tissuesusing standard methodologies and minor modifications thereof in tissueswhere expression may be low. For example, protein distribution may bedetermined using standard Western blot analysis or immunofluorescenttechniques, and antibodies specific to the morphogen or morphogens ofinterest. Similarly, the distribution of morphogen transcripts may bedetermined using standard Northern hybridization protocols andtranscript-specific probes.

[0100] Any probe capable of hybridizing specifically to a transcript,and distinguishing the transcript of interest from other, relatedtranscripts may be used. Because the morphogens described herein sharesuch high sequence homology in their active, C-terminal domains, thetissue distribution of a specific morphogen transcript may best bedetermined using a probe specific for the pro region of the immatureprotein and/or the N-terminal region of the mature protein. Anotheruseful sequence is the 3′ non-coding region flanking and immediatelyfollowing the stop codon. These portions of the sequence varysubstantially among the morphogens of this invention, and accordingly,are specific for each protein. For example, a particularly usefulVgr-1-specific probe sequence is the PvuII-SacI fragment, a 265 bpfragment encoding both a portion of the untranslated pro region and theN-terminus of the mature sequence. See Lyons, et al., PNAS 86: 4554-4558(1989) for a description of the cDNA sequence. Similarly, particularlyuseful mOP-1-specific probe sequences are the BstX1-BglI fragment, a0.68 Kb sequence that covers approximately two-thirds of the mOP-1 proregion; a StuI-StuI fragment, a 0.2 Kb sequence immediately upstream ofthe 7-cysteine domain; and the Earl -Pst1 fragment, an 0.3 Kb fragmentcontaining a portion of the 3′ untranslated sequence (See SEQ ID NO:18,where the pro region is defined essentially by residues 30-291.) Similarapproaches may be used, for example, with hOP1 (SEQ ID NO:16) or humanor mouse OP-2 (SEQ ID NOS:20 and 22.)

[0101] Using these morphogen-specific probes, which may be syntheticallyengineered or obtained from cloned sequences, morphogen transcripts canbe identified in mammalian tissue, using standard methodologies wellknown to those having ordinary skill in the art. Briefly, total RNA isprepared from various adult murine tissues (e.g., liver, kidney, testis,heart, brain, thymus and stomach) by a standard methodology such as bythe method of Chomczyaski, et al., Anal. Biochem 162: 156-159 (1987) anddescribed below. Poly (A)+ RNA is prepared by using oligo (dT)-cellulosechromatography (e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.).Poly (A)+RNA (generally 15 mg) from each tissue is fractionated on a 1%agarose/formaldehyde gel and transferred onto a Nytran membrane(Schleicher & Schuell). Following the transfer, the membrane is baked at80° C. and the RNA is cross-linked under UV light (generally 30 secondsat 1 mW/cm²). Prior to hybridization, the appropriate probe is denaturedby heating. The hybridization is carried out in a lucite cylinderrotating in a roller bottle apparatus at approximately 1 rev/min forapproximately 15 hours at 37° C. using a hybridization mix of 40%formamide, 5×Denhardts, 5×SSPE, and 0.1% SDS. Following hybridization,the non-specific counts are washed off the filters in 0.1×SSPE, 0.1% SDSat 50° C.

[0102] Examples demonstrating the tissue distribution of variousmorphogens, including Vgr-1, OP-1, BMP2, BMP3, BMP4, BMP5, GDF-1, andOP-2 in developing and adult tissue are disclosed in co-pending U.S.Ser. No. 752,764, and in Ozkaynak, et al., Biochem. Biophys. Res. Comm.179: 116-123 (1991), and Ozkaynak, et al., (JBC, in press) (1992), thedisclosures of which are incorporated herein by reference. Using thegeneral probing methodology described herein, northern blothybridizations using probes specific for these morphogens to probebrain, spleen, lung, heart, liver and kidney tissue indicate thatkidney-related tissue appears to be the primary expression source forOP-1, with brain, heart and lung tissues being secondary sources. Lungtissue appears to be the primary tissue expression source for Vgr-1,BMP5, BMP4 and BMP3. Lower levels of Vgr-1 also are seen in kidney andheart tissue, while the liver appears to be a secondary expressionsource for BMP5, and the spleen appears to be a secondary expressionsource for BMP4. GDF-1 appears to be expressed primarily in braintissue. To date, OP-2 appears to be expressed primarily in earlyembryonic tissue. Specifically, northern blots of murine embryos and6-day post-natal animals shows abundant OP2 expression in 8-day embryos.Expression is reduced significantly in 17-day embryos and is notdetected in post-natal animals.

EXAMPLE 3 Morphogen Localization in the Nervous System

[0103] Morphogens have been identified in developing and adult rat brainand spinal cord tissue, as determined both by northern blothybridization of morphogen-specific probes to mRNA extracts fromdeveloping and adult nerve tissue (see Example 2, above) and byimmunolocalization studies. For example, northern blot analysis ofdeveloping rat tissue has identified significant OP-1 mRNA transcriptexpression in the CNS (U.S. Ser. No. 752,764, and Ozkaynak, et al.,Biochem. Biophys. Res. Comm., 179: 11623 (1991) and Ozkaynak, et al.,JBC, in press (1992)). GDF-1 mRNA appears to be expressed primarily indeveloping and adult nerve tissue, specifically in the brain, includingthe cerebellum and brain stem, spinal cord and peripheral nerves. Lee,S., PNAS 88: 4250-4254 (1991). BMP2B (also referred in the art as BMP4)and Vgr-1 transcripts also have been reported to be expressed in nervetissue (Jones, et al., Development 111: 531-542 (1991)), although thenerve tissue does not appear to be the primary expression tissue forthese genes. Ozkaynak, et al., JBC in press (1992). Specifically, CBMP2transcripts are reported in the region of the diencephalon associatedwith pituitary development, and Vgr-1 transcripts are reported in theanteroposterior axis of the CNS, including in the roof plate of thedeveloping neural tube, as well as in the cells immediately adjacent thefloor plate of the developing neural tube. In older rats, Vgr-1transcripts are reported in developing hippocampus tissue. In addition,the genes encoding OP-1 and BMP2 originally were identified by probinghuman hippocampus cDNA libraries.

[0104] Immunolocalization studies, performed using standardmethodologies known in the art and disclosed in U.S. Ser. No. 752,764,filed Aug. 30, 1991, the disclosure of which is incorporated herein,localized OP-1 expression to particular areas of developing and adultrat brain and spinal cord tissue. Specifically, OP1 protein expressionwas assessed in adult (2-3 months old) and five or six-day old mouseembryonic nerve tissue, using standard morphogen-specific antisera,specifically, rabbit anti-OP1 antisera, made using standard antibodyprotocols known in the art and preferably purified on an OP1 affinitycolumn. The antibody itself was labelled using standard fluorescentlabelling techniques, or a labelled anti-rabbit IgG molecule was used tovisualize bound OP-1 antibody.

[0105] As can be seen in FIG. 2, immunofluorescence stainingdemonstrates the presence of OP-1 in adult rat central nervous system(CNS.) Similar and extensive staining is seen in both the brain (Panel1A) and spinal cord (Panel 1B). OP-1 appears to be localizedpredominantly to the extracellular matrix of the grey matter (neuronalcell bodies), distinctly present in all areas except the cell bodiesthemselves. In white matter, formed mainly of myelinated nerve fibers,staining appears to be confined to astrocytes (glial cells). A similarstaining pattern also was seen in newborn rat (10 day old) brainsections.

[0106] In addition, OP-1 has been specifically localized in thesubstantia nigra, which is composed primarily of striatal basal ganglia,a system of accessory motor neurons that function is association withthe cerebral cortex and corticospinal system. Dysfunctions in thissubpopulation or system of neurons are associated with a number ofneuropathies, including Huntington's chorea and Parkinson's disease.

[0107] OP-1 also has been localized at adendema glial cells, known tosecrete factors into the cerebrospinal fluid, and which occur around theintraventricular valve, coroid fissure, and central canal of the brainin both developing and adult rat.

[0108] Finally, morphogen inhibition in developing embryos inhibitsnerve tissue development. Specifically, 9-day mouse embryo cells,cultured in vitro under standard culturing conditions, were incubated inthe presence and absence of an OP-1-specific monoclonal antibodyprepared using recombinantly produced, purified mature OP1 and theimmunogen. The antibody was prepared using standard antibody productionmeans well known in the art and as described generally in Example 14.After two days, the effect of the antibody on the developing embryo wasevaluated by histology. As determined by histological examination, theOP-1-specific antibody specifically inhibits eye lobe formation in thedeveloping embryo. In particular, the diencephalon outgrowth does notdevelop. In addition, the heart is malformed and enlarged. Moreover, inseparate immunolocalization studies on embryo sections with labelledOP-1 specific antibody, the OP-1 -specific antibody localizes to neuralepithelia.

[0109] The endogenous morphogens which act on neuronal cells may beexpressed and secreted by nerve tissue cells, e.g., by neurons and/orglial cells associated with the neurons, and/or they may be transportedto the neurons by the cerebrospinal fluid and/or bloodstream. Recently,OP-1 has been identified in the human blood (See Example 10, below). Inaddition, transplanted Schwann cells recently have been shown tostimulate nerve fiber formation in rat spinal cord, including inducingvascularization and myelin sheath formation around at least some of thenew neuronal processes. Bunge, Exp. Neurology 114: 254-257 (1991). Theregenerative property of these cells may be mediated by the secretion ofa morphogen by the Schwann cells.

EXAMPLE 4 Morphogen Enhancement of Neuronal Cell Survival

[0110] The morphogens described herein enhance cell survival,particularly of neuronal cells at risk of dying. For example, fullydifferentiated neurons are non-mitotic and die in vitro when culturedunder standard mammalian cell culture conditions, using a chemicallydefined or low serum medium known in the art. See, for example,Charness, J. Biol. Chem. 26: 3164-3169 (1986) and Freese, et al., BrainRes. 521: 254-264 (1990). However, if a primary culture of non-mitoticneuronal cells is treated with a morphogen, the survival of these cellsis enhanced significantly. For example, a primary culture of striatalbasal ganglia isolated from the substantia nigra of adult rat brain wasprepared using standard procedures, e.g., by dissociation by triturationwith pasteur pipette of substantia nigra tissue, using standard tissueculturing protocols, and grown in a low serum medium, e.g., containing50% DMEM (Dulbecco's modified Eagle's medium), 50% F-12 medium, heatinactivated horse serum supplemented with penicillin/streptomycin and 4g/l glucose. Under standard culture conditions, these cells areundergoing significant cell death by three weeks when cultured in aserum-free medium. Cell death is evidenced morphologically by theinability of cells to remain adherent and by changes in theirultrastructural characteristics, e.g., by chromatin clumping andorganelle disintegration.

[0111] In this example, the cultured basal ganglia were treated withchemically defined medium conditioned with 0.1-100 ng/ml OP-1. Fresh,morphogen-conditioned medium was provided to the cells every 3-4 days.Cell survival was enhanced significantly and was dose dependent upon thelevel of OP1 added: cell death decreased significantly as theconcentration of OP1 was increased in cell cultures. Specifically, cellsremained adherent and continued to maintain the morphology of viabledifferentiated neurons. In the absence of morphogen treatment, themajority of the cultured cells dissociated and underwent cell necrosis.

[0112] Dysfunctions in the basal ganglia of the substantia nigra areassociated with Huntington's chorea and parkinsonism in vivo. Theability of the morphogens defined herein to enhance neuron survivalindicates that these morphogens will be useful as part of a therapy toenhance survival of neuronal cells at risk of dying in vivo due, forexample, to a neuropathy or chemical or mechanical trauma. It isparticularly anticipated that these morphogens will provide a usefultherapeutic agent to treat neuropathies which affect the striatal basalganglia, including Huntington's chorea and Parkinson's disease. Forclinical applications, the morphogen may be administered or,alternatively, a morphogen-stimulating agent may be administered.

EXAMPLE 5 Morphogen-Induced Redifferentiation of Transformed Cells

[0113] The morphogens described herein also induce redifferentiation oftransformed cells to a morphology characteristic of untransformed cells.In particular, the morphogens are capable of inducing redifferentiationof transformed cells of neuronal origin to a morphology characteristicof untransformed neurons. The example provided below details morphogeninduced redifferentiation of a transformed cell line of neuronal origin,NG105-115. Morphogen-induced redifferentiation of transformed cells alsohas been shown in mouse neuroblastoma cells (N1E-115) and in humanembryo carcinoma cells (see copending U.S. Ser. No. 752,764,incorporated herein by reference.)

[0114] NG108-15 is a transformed hybrid cell line produced by fusingneuroblastoma× glioma cells (obtained from America Type Tissue Culture,Rockville, Md.), and exhibiting a morphology characteristic oftransformed embryonic neurons, e.g., having a fibroblastic morphology.Specifically, the cells have polygonal cell bodies, short, spike-likeprocesses and make few contacts with neighboring cells (see FIG. 2A).Incubation of NG1 08-15 cells, cultured in a chemically defined,serum-free medium, with 0.1 to 300 ng/ml of OP-1 for four hours inducesan orderly, dose-dependent change in cell morphology.

[0115] In the experiment NG108-15 cells were subcultured onpoly-L-lysine coated 6-well plates. Each well contained 40-50,000 cellsin 2.5 ml of chemically defined medium. On the third day 2.5 ml of OP-1in 60% ethanol containing 0.025% trifluoroacetic was added to each well.OP-1 concentrations of 0-300 ng/ml were tested. Typically, the media waschanged daily with new aliquots of OP-1, although morphogenesis can beinduced by a single four hour incubation with OP-1. In addition, OP-1concentrations of 10 ng/ml were sufficient to induce redifferentiation.After one day, hOP-1-treated cells undergo a significant change in theircellular ultrastructure, including rounding of the soma, increase inphase brightness and extension of the short neurite processes. After twodays, cells treated with OP-1 begin to form epithelioid sheets, whichprovide the basis for the growth of mutilayered aggregates at threeday's post-treatment. By four days, the great majority of OP-1-treatedcells are associated in tightly-packed, mutilayered aggregates (FIG.2B). FIG. 4 plots the mean number of multi-layered aggregates or cellclumps identified in twenty randomly selected fields from sixindependent experiments, as a function of morphogen concentration. Fortyng/ml of OP-1 is sufficient for half maximum induction of cellaggregation.

[0116] The morphogen-induced redifferentiation occurred without anyassociated changes in DNA synthesis, cell division, or cell viability,making it unlikely that the morphologic changes were secondary to celldifferentiation or a toxic effect of hOP-1. Moreover, the OP1-inducedmorphogenesis does not inhibit cell division, as determined by³H-thymidine uptake, unlike other molecules which have been shown tostimulate differentiation of transformed cells, such as butyrate, DMSO,retinoic acid or Forskolin. The data indicate that OP-1 can maintaincell stability and viability after inducing redifferentiation. Inaddition, the effects are morphogen specific, and redifferentiation isnot induced when NG108-15 cells are incubated with 0.1-40 ng/ml TGF-β.

[0117] The experiments also have been performed with highly purifiedsoluble morphogen (e.g., mature OP-1 associated with its pro domain)which also specifically induced redifferentiation of NG108-15 cells.

[0118] The morphogens described herein accordingly provide usefultherapeutic agents for the treatment of neoplasias and neoplasticlesions of the nervous system, particularly in the treatment ofneuroblastomas, including retinoblastomas, and gliomas. The morphogensthemselves may be administered or, alternatively, amorphogen-stimulating agent may be administered.

EXAMPLE 6 Nerve Tissue Protection from Chemical Trauma

[0119] The ability of the morphogens described herein to enhancesurvival of neuronal cells and to induce cell aggregation and cell-celladhesion in redifferentiated cells, indicates that the morphogens willbe useful as therapeutic agents to maintain neural pathways byprotecting the cells defining the pathway from the damage caused bychemical trauma. In particular, the morphogens can protect neurons,including developing neurons, from the effects of toxins known toinhibit the proliferation and migration of neurons and to interfere withcell-cell adhesion. Examples of such toxins include ethanol, one or moreof the toxins present in cigarette smoke, and a variety of opiates. Thetoxic effects of ethanol on developing neurons induces the neurologicaldamage manifested in fetal alcohol syndrome. The morphogens also mayprotect neurons from the cytotoxic effects associated with excitatoryamino acids such as glutamate.

[0120] For example, ethanol inhibits the cell-cell adhesion effectsinduced in morphogen-treated NG108-15 cells when provided to these cellsat a concentration of 25-50 mM. Half maximal inhibition can be achievedwith 5-10 mM ethanol, the concentration of blood alcohol in an adultfollowing ingestion of a single alcoholic beverage. Ethanol likelyinterferes with the homophilic binding of CAMs between cells, ratherthan their induction, as morphogen-induced N-CAM levels are unaffectedby ethanol. Moreover, the inhibitory effect is inversely proportional tomorphogen concentration. Accordingly, it is envisioned thatadministration of a morphogen or morphogen-stimulating agent to neurons,particularly developing neurons, at risk of damage from exposure totoxins such as ethanol, may protect these cells from nerve tissue damageby overcoming the toxin's inhibitory effects. The morphogens describedherein also are useful in therapies to treat damaged neural pathwaysresulting from a neuropathy induced by exposure to these toxins.

EXAMPLE 7 Morphogen-Induced CAM Expression

[0121] The morphogens described herein induce CAM expression,particularly N-CAM expression, as part of their induction ofmorphogenesis. CAMs are morphoregulatory molecules identified in alltissues as an essential step in tissue development. N-CAMs, whichcomprise at least 3 isoforms (N-CAM-180, N-CAM-140 and N-CAM-120, where“180”, “140” and “120” indicate the apparent molecular weights of theisoforms as measured by polyacrylamide gel electrophoresis) areexpressed at least transiently in developing tissues, and permanently innerve tissue. Both the N-CAM-180 and N-CAM-140 isoforms are expressed inboth developing and adult tissue. The N-CAM-120 isoform is found only inadult tissue. Another neural CAM is L 1.

[0122] N-CAMs are implicated in appropriate neural development,including appropriate neurulation, neuronal migration, fasciculation,and synaptogenesis. Inhibition of N-CAM production, as by complexing themolecule with an N-CAM-specific antibody, inhibits retina organization,including retinal axon migration, and axon regeneration in theperipheral nervous system, as well as axon synapses with target musclecells. In addition, significant evidence indicates that physical orchemical trauma to neurons, oncogenic transformation and some geneticneurological disorders are accompanied by changes in CAM expression,which alter the adhesive or migratory behavior of these cells.Specifically, increased N-CAM levels are reported in Huntington'sdisease striatum (e.g., striatal basal ganglia), and decreased adhesionis noted in Alzheimer's disease.

[0123] The morphogens described herein stimulate CAM production,particularly L1 and N-CAM production, including all three isoforms ofthe N-CAM molecule. For example, N-CAM expression is stimulatedsignificantly in morphogen-treated NG108-15 cells. Untreated NG108-15cells exhibit a fibroblastic, or minimally differentiated morphology andexpress only the 180 and 140 isoforms of N-CAM normally associated witha developing cell. Following morphogen treatment, these cells exhibit amorphology characteristic of adult neurons and express enhanced levelsof all three N-CAM isoforms. Using a similar protocol as described inthe example below, morphogen treatment of NG108-15 cells also induced L1expression.

[0124] In this example, NG108-15 cells were cultured for four days inthe presence of increasing concentrations of OP-1 and standard Westernblots performed on whole cell extracts. N-CAM isoforms were detectedwith an antibody which cross-reacts with all three isoforms, mAbH28.123, obtained from Sigma Chemical Co., St. Louis, the differentisoforms being distinguishable by their different mobilities on anelectrophoresis gel. Control NG108-15 cells (untreated) express both the140 kDa and the 180 kDa isoforms, but not the 120 kDa, as determined bywestern blot analyses using up to 100 mg of protein. Treatment ofNG108-15 cells with OP-1 resulted in a dose-dependent increase in theexpression of the 180 kDa and 140 kDa isoforms, as well as the inductionof the 120 kDa isoform. See FIG. 3. FIG. 3B is a Western blot ofOP1-treated NG108-15 cell extracts, probed with mAb H28.123, showing theinduction of all three isoforms. FIG. 3A is a dose response curve ofN-CAM-180 and N-CAM-140 induction as a function of morphogenconcentration. N-CAM-120 is not shown in the graph, as it could not bequantitated in control cells. However, as is clearly evident from theWestern blot in FIG. 3B, N-CAM-120 is induced in response to morphogentreatment.

[0125] The increase in N-CAM expression corresponded in a dose-dependentmanner with the morphogen induction of multicellular aggregates. CompareFIG. 3A and FIG. 4. FIG. 4 graphs the mean number of multilayeredaggregates (clumps) counted per 20 randomly selected, microscopicviewing fields in six independent experiments, versus the concentrationof morphogen. The induction of the 120 isoform also indicates thatmorphogen-induced redifferentiation of transformed cells stimulates notonly redifferentiation of these cells from a transformed phenotype, butalso differentiation to a phenotype corresponding to a developed cell.Standard immunolocalization studies performed with the mAb H28.123 onmorphogen-treated cells show N-CAM cluster formation associated with theperiphery and processes of treated cells, and no reactivity withuntreated cells. Moreover, morphogen treatment does not appear toinhibit cell division as determined by cell counting or ³H-thymidineuptake. Finally, known chemical differentiating agents, such asForskolin and dimethylsulfoxide, do not induce N-CAM production.

[0126] In addition, the cell aggregation effects of OP1 on NG108-15cells can be inhibited with anti-N-CAM antibodies or antisense N-CAMoligonucleotides. Antisense oligonucleotides can be made syntheticallyon a nucleotide synthesizer, using standard means known in the art.Preferably, phosphorothioate oligonucleotides (“S-oligos”) are prepared,to enhance transport of the nucleotides across cell membranes.Concentrations of both N-CAM antibodies and N-CAM antisenseoligonucleotides sufficient to inhibit N-CAM induction also inhibitedformation of multilayered cell aggregates. Specifically, incubation ofmorphogen-treated NG108-15 cells with 0.3-3 mM N-CAM antisense S-oligos,5-500 mM unmodified N-CAM antisense oligos, or 10 mg/ml mAb H28.123significantly inhibits cell aggregation. It is likely that morphogentreatment also stimulates other CAMs, as inhibition is not complete.

[0127] Finally, the above-described experiments have also been performedwith soluble morphogen (e.g., mature OP1 dimer, associated with its prodomain polypeptides as described in Example 1). The soluble form ofmorphogen also specifically induced CAM expression.

[0128] In addition to a transformed cell line, N-CAM expression can beassayed in a primary cell culture of neural or glial cells, followingthe procedures described herein. The efficacy of the morphogensdescribed herein to affect N-CAM expression can be assessed in vitrousing a suitable cell line, such as NG108-15 and the methods describedherein.

[0129] As described above, preferred morphogens, inducers, or agonistsof the present invention can induce both N-CAM expression in vitro andendochondral bone formation when implanted in vivo in a mammal inconjunction with a matrix permissive of bone morphogenesis. Thus, themethods described herein can be used to assess novel candidatemorphogens, inducers, or agonists.

[0130] The experiments also have been performed with soluble morphogen(e.g., mature OP-1 associated with its pro domain) which alsospecifically induced CAM expression.

[0131] The morphogens described herein are useful as therapeutic agentsto treat neurological disorders associated with altered CAM levels,particularly N-CAM levels, such as Huntington's chorea and Alzheimer'sdisease, and the like. In clinical applications, the morphogensthemselves may be administered or, alternatively, amorphogen-stimulating agent may be administered.

[0132] The efficacy of the morphogens described herein to affect N-CAMexpression may be assessed in vitro using a suitable cell line and themethods described herein. In addition to a transformed cell line, N-CAMexpression can be assayed in a primary cell culture of neural or glialcells, following the procedures described herein. The efficacy ofmorphogen treatment on N-CAM expression in vivo may be evaluated bytissue biopsy as described in Example 10, below, and detecting N-CAMmolecules with an N-CAM-specific antibody, such as mAb H28.123, or usingthe animal model described in Example 12.

[0133] Alternatively, the level of N-CAM proteins or protein fragmentspresent in cerebrospinal fluid or serum also may be detected to evaluatethe effect of morphogen treatment. N-CAM molecules are known to sloughoff cell surfaces and have been detected in both serum and cerebrospinalfluid. In addition, altered levels of the soluble form of N-CAM areassociated with normal pressure hydrocephalus and type II schizophrenia.N-CAM fluid levels may be detected following the procedure described inExample 10 and using an N-CAM specific antibody, such as mAb H28.123.

EXAMPLE 8 Morphogen-Induced Nerve Gap Repair (PNS)

[0134] The morphogens described herein also stimulate peripheral nervoussystem axonal growth over extended distances allowing repair andregeneration of damaged neural pathways. While neurons of the peripheralnervous system can sprout new processes following injury, withoutguidance these sproutings typically fail to connect appropriately anddie. Where the break is extensive, e.g., greater than 5 or 10 mm,regeneration is poor or nonexistent.

[0135] In this example morphogen stimulation of nerve regeneration wasassessed using the rat sciatic nerve model. The rat sciatic nerve canregenerate spontaneously across a 5 mm gap, and occasionally across a 10mm gap, provided that the severed ends are inserted in a saline-fillednerve guidance channel. In this experiment, nerve regeneration across a12 mm gap was tested.

[0136] Adult female Sprague-Dawley rats (Charles River Laboratories,Inc.) weighing 230-250 g were anesthetized with intraperitonealinjections of sodium pentobarbital 35 mg/kg body weight). A skinincision was made parallel and just posterior to the femur. Theavascular intermuscular plane between vastus lateralis and hamstringmuscles were entered and followed to the loose fibroareolar tissuesurrounding the sciatic nerve. The loose tissue was dividedlongitudinally thereby freeing the sciatic nerve over its full extentwithout devascularizing any portion. Under a surgical microscope thesciatic nerves were transected with microscissors at mid-thigh andgrafted with an OP-1 gel graft that separated the nerve stumps by 12 mm.The graft region was encased in a silicone tube 20 mm in length with a1.5 mm inner diameter, the interior of which was filled a morphogensolution. Specifically, The central 12 mm of the tube consisted of anOP-1 gel prepared by mixing 1 to 5 mg of substantially pure CHO-producedrecombinant OP-1 with approximately 100 ml of MATRIGEL™ (fromCollaborative Research, Inc., Bedford, Mass.), an extracellular matrixextract derived from mouse sarcoma tissue, and containing solubilizedtissue basement membrane, including laminin, type IV collagen, heparinsulfate, proteoglycan and entactin, in phosphate-buffered saline. TheOP-1-filled tube was implanted directly into the defect site, allowing 4mm on each end to insert the nerve stumps. Each stump was abuttedagainst the OP-1 gel and was secured in the silicone tube by threestitches of commercially available surgical 10-0 nylon through theepineurium, the fascicle sheath.

[0137] In addition to OP-1 gel grafts, empty silicone tubes, siliconetubes filled with gel only and “reverse” autografts, wherein 12 mmtransected segments of the animal's sciatic nerve were rotated 180°prior to suturing, were grafted as controls. All experiments wereperformed in quadruplicate. All wounds were closed by wound clips thatwere removed after 10 days. All rats were grafted on both legs. At 3weeks the animals were sacrificed, and the grafted segments removed andfrozen on dry ice immediately. Frozen sections then were cut throughoutthe graft site, and examined for axonal regeneration byimmunofluorescent staining using anti-neurofilament antibodies labeledwith flurocein (obtained from Sigma Chemical Co., St. Louis).

[0138] Regeneration of the sciatic nerve occurred across the entire 12mm distance in all graft sites wherein the gap was filled with the OP-1gel. By contrast, empty silicone tubes and reverse autografts did notshow nerve regeneration, and only one graft site containing the gelalone showed axon regeneration.

EXAMPLE 9 Morphogen-Induced Nerve Gap Repair (CNS)

[0139] Following axonal damage in vivo the CNS neurons are unable toresprout processes. Accordingly, trauma to CNS nerve tissue, includingthe spinal cord, optic nerve and retina, severely damages or destroysthe neural pathways defined by these cells. Peripheral nerve grafts havebeen used in an effort to bypass CNS axonal damage. Successfulautologous graft repair to date apparently requires that the graft siteoccur near the CNS neuronal cell body, and a primary result of CNSaxotomy is neuronal cell death. The efficacy of morphogens describedherein on CNS nerve repair, may be evaluated using a rat crushed opticnerve model such as the one described by Bignami, et al., Exp. Eye Res.28: 63-69 (1979), the disclosure of which is incorporated herein byreference. Briefly, and as described therein, laboratory rats (e.g.,from Charles River Laboratories, Wilmington, Mass.) are anesthetizedusing standard surgical procedures, and the optic nerve crushed bypulling the eye gently out of the orbit, inserting a watchmaker forcepsbehind the eyeball and squeezing the optic nerve with the forceps for 15seconds, followed by a 30 second interval and second 15 second squeeze.Rats are sacrificed at different time intervals, e.g., at 48 hours, andat 3, 4, 11, 15 and 18 days after operation. The effect of morphogen onoptic nerve repair can be assessed by performing the experiment induplicate and providing morphogen or PBS (e.g., 25 ml solution, and 25mg morphogen) to the optic nerve, e.g., just prior to the operation,concomitant with the operation, or at specific times after theoperation.

[0140] In the absence of therapy, the surgery induces glial scarring ofthe crushed nerve, as determined by immunofluorescence staining forglial fibrillary acidic protein (GFA), a marker protein for glialscarring, and by histology. Indirect immunofluorescence on air-driedcryostat sections as described in Bignami, et al., J. Comp. Neur. 153:27-38 (1974), using commercially available antibodies to GFA (e.g.,Sigma Chemical Co., St. Louis). Reduced levels of GFA are anticipated inanimals treated with the morphogen, evidencing the ability of morphogensto inhibit glial scar formation and to stimulate optic nerveregeneration.

EXAMPLE 10 Nerve Tissue Diagnostics

[0141] Morphogen localization in nerve tissue can be used as part of amethod for diagnosing a neurological disorder or neuropathy. The methodmay be particularly advantageous for diagnosing neuropathies of braintissue. Specifically, a biopsy of brain tissue is performed on a patientat risk, using standard procedures known in the medical art. Morphogenexpression associated with the biopsied tissue then is assessed usingstandard methodologies, as by immunolocalization, using standardimmunofluorescence techniques in concert with morphogen-specificantisera or monoclonal antibodies. Specifically, the biopsied tissue isthin sectioned using standard methodologies known in the art, andfluorescently labelled (or otherwise detectable) antibodies incubatedwith the tissue under conditions sufficient to allow specificantigen-antibody complex formation. The presence and quantity of complexformed then is detected and compared with a predetermined standard orreference value. Detection of altered levels of morphogen present in thetissue then may be used as an indicator of tissue dysfunction.Alternatively, fluctuation in morphogen levels may be assessed bymonitoring morphogen transcription levels, either by standard northernblot analysis or in situ hybridization, using a labelled probe capableof hybridizing specifically to morphogen RNA and standard RNAhybridization protocols well described in the art.

[0142] Fluctuations in morphogen levels present in the cerebrospinalfluid or bloodstream also may be used to evaluate nerve tissueviability. For example, morphogens are detected associated with adendemacells which are known to secrete factors into the cerebrospinal fluid,and are localized generally associated with glial cells, and in theextracellular matrix, but not with neuronal cell bodies. Accordingly,the cerebrospinal fluid may be a natural means of morphogen transport.Alternatively, morphogens may be released from dying cells intocerebrospinal fluid. In addition, OP-1 recently has been identified inhuman blood, which also may be a means of morphogen transport, and/or arepository for the contents of dying cells.

[0143] Spinal fluid may be obtained from an individual by a standardlumbar puncture, using standard methodologies known in the medical art.Similarly, serum samples may be obtained by standard venipuncture andserum prepared by centrifugation at 3,000 RPM for ten minutes. Thepresence of morphogen in the serum or cerebral spinal fluid then may beassessed by standard Western blot (immunoblot), ELISA or RIA procedures.Briefly, for example, with the ELISA, samples may be diluted in anappropriate buffer, such as phosphate-buffered saline, and 50 mlaliquots allowed to absorb to flat bottomed wells in microtitre platespre-coated with morphogen-specific antibody, and allowed to incubate for18 hours at 4° C. Plates then may be washed with a standard buffer andincubated with 50 ml aliquots of a second morphogen-specific antibodyconjugated with a detecting agent, e.g., biotin, in an appropriatebuffer, for 90 minutes at room temperature. Morphogen-antibody complexesthen may be detected using standard procedures.

[0144] Alternatively, a morphogen-specific affinity column may becreated using, for example, morphogen-specific antibodies adsorbed to acolumn matrix, and passing the fluid sample through the matrix toselectively extract the morphogen of interest. The morphogen then iseluted. A suitable elution buffer may be determined empirically bydetermining appropriate binding and elution conditions first with acontrol (e.g., purified, recombinantly-produced morphogen.) Fractionsthen are tested for the presence of the morphogen by standardimmunoblot, and confirmed by N-terminal sequencing. Morphogenconcentrations in serum or other fluid samples then may be determinedusing standard protein quantification techniques, including byspectrophotometric absorbance or by quantitation by ELISA or RIAantibody assays. Using this procedure, OP-1 has been identified inserum.

[0145] OP1 was detected in human serum using the following assay. Amonoclonal antibody raised against mammalian, recombinantly producedOP-1 using standard immunology techniques well described in the art anddescribed generally in Example 14, was immobilized by passing theantibody over an activated agarose gel (e.g., Affi-Gel™, from Bio-RadLaboratories, Richmond, Calif., prepared following manufacturer'sinstructions), and used to purify OP-1 from serum. Human serum then waspassed over the column and eluted with 3M K-thiocyanate. K-thiocyanantefractions then were dialyzed in 6M urea, 20 mM PO₄, pH 7.0, applied to aC8 HPLC column, and eluted with a 20 minute, 25-50% acetonitrile/0.1%TFA gradient. Mature, recombinantly produced OP-1 homodimers elutebetween 20-22 minutes. Fractions then were collected and tested for thepresence of OP1 by standard immunoblot. FIG. 5 is an immunoblot showingOP1 in human sera under reducing and oxidized conditions. In the figure,lanes 1 and 4 are OP1 standards, run under oxidized (lane 1) and reduced(lane 4) conditions. Lane 5 shows molecular weight markers at 17, 27 and39 kDa. Lanes 2 and 3 are human sera OP-1, run under oxidized (lane 2)and reduced (lane 3) conditions.

[0146] Morphogens may be used in diagnostic applications by comparingthe quantity of morphogen present in a body fluid sample with apredetermined reference value, with fluctuations in fluid morphogenlevels indicating a change in the status of nerve tissue. Alternatively,fluctuations in the level of endogenous morphogen antibodies may bedetected by this method, most likely in serum, using an antibody orother binding protein capable of interacting specifically with theendogenous morphogen antibody. Detected fluctuations in the levels ofthe endogenous antibody may be used as indicators of a change in tissuestatus.

EXAMPLE 11 Alleviation of Immune Response-Mediated Nerve Tissue Damage

[0147] The morphogens described herein may be used to alleviateimmunologically-related damage to nerve tissue. Details of this damageand the use of morphogens to alleviate this injury are disclosed incopending U.S. Ser. No. 753,059, filed Aug. 30, 1991, the disclosure ofwhich is incorporated herein. A primary source of such damage to nervetissue follows hypoxia or ischemia-reperfusion of a blood supply to aneural pathway, such as may result from an embolic stroke, or may beinduced during a surgical procedure. As described in U.S. Ser. No.753,059, morphogens have been shown to alleviate damage to myocardialtissue following ischemia-reperfusion of the blood supply to the tissue.The effect of morphogens on alleviating immunologically-related damageto nerve tissue may be assessed using methodologies and models known tothose skilled in the art and described below.

[0148] For example, the rabbit embolic stroke model provides a usefulmethod for assessing the effect of morphogens on tissue injury followingcerebral ischemia-reperfusion. The protocol disclosed below isessentially that of Phillips, et al., Annals of Neurology 25: 281-285(1989), the disclosure of which is herein incorporated by reference.Briefly, white New England rabbits (2-3 kg) are anesthetized and placedon a respirator. The intracranial circulation then is selectivelycatheterized by the Seldinger technique. Baseline cerebral angiographythen is performed, employing a digital substration unit. The distalinternal carotid artery or its branches then is selectively embolizedwith 0.035 ml of 18-hour-aged autologous thrombus. Arterial occlusion isdocumented by repeat angiography immediately after embolization. After atime sufficient to induce cerebral infarcts (15 minutes or 90 minutes),reperfusion is induced by administering a bolus of a reperfusion agentsuch as the TPA analogue FB-FB-CF (e.g., 0.8 mg/kg over 2 minutes).

[0149] The effect of morphogen on cerebral infarcts can be assessed byadministering varying concentrations of morphogens, e.g., OP-1, atdifferent times following embolization and/or reperfusion. The rabbitsare sacrificed 3-14 days post embolization and their brains prepared forneuropathological examination by fixing by immersion in 10% neutralbuffered formation for at least 2 weeks. The brains then are sectionedin a coronal plane at 2-3 mm intervals, numbered and submitted forstandard histological processing in paraffin, and the degree of nervetissue necrosis determined visually. Morphogen-treated animals areanticipated to reduce or significantly inhibit nerve tissue necrosisfollowing cerebral ischemia-reperfusion in the test animals asdetermined by histology comparison with non-treated animals.

EXAMPLE 12 Animal Model for Assessing Morphogen Efficacy In Vivo

[0150] The in vivo activities of the morphogens described herein alsoare assessed readily in an animal model as described herein. A suitableanimal, preferably exhibiting nerve tissue damage, for example,genetically or environmentally induced, is injected intracerebrally withan effective amount of a morphogen in a suitable therapeuticformulation, such as phosphate-buffered saline, pH 7. The morphogenpreferably is injected within the area of the affected neurons. Theaffected tissue is excised at a subsequent time point and the tissueevaluated morphologically and/or by evaluation of an appropriatebiochemical marker (e.g., by morphogen or N-CAM localization; or bymeasuring the dose-dependent effect on a biochemical marker for CNSneurotrophic activity or for CNS tissue damage, using for example, glialfibrillary acidic protein as the marker. The dosage and incubation timewill vary with the animal to be tested. Suitable dosage ranges fordifferent species may be determined by comparison with establishedanimal models. Presented below is an exemplary protocol for a rat brainstab model.

[0151] Briefly, male Long Evans rats, obtained from standard commercialsources, are anesthetized and the head area prepared for surgery. Thecalvariae is exposed using standard surgical procedures and a holedrilled toward the center of each lobe using a 0.035K wire, justpiercing the calvariae. 25 ml solutions containing either morphogen(e.g., OP-1, 25 mg) or PBS then is provided to each of the holes byHamilton syringe. Solutions are delivered to a depth approximately 3 mmbelow the surface, into the underlying cortex, corpus callosum andhippocampus. The skin then is sutured and the animal allowed to recover.

[0152] Three days post surgery, rats are sacrificed by decapitation andtheir brains processed for sectioning. Scar tissue formation isevaluated by immunofluorescence staining for glial fibrillary acidicprotein, a marker protein for glial scarring, to qualitatively determinethe degree of scar formation. Glial fibrillary acidic protein antibodiesare available commercially, e.g., from Sigma Chemical Co., St. Louis,Mo. Sections also are probed with anti-OP-1 antibodies to determine thepresence of OP-1. Reduced levels of glial fibrillary acidic protein areanticipated in the tissue sections of animals treated with themorphogen, evidencing the ability of morphogens to inhibit glial scarformation and stimulate nerve regeneration.

EXAMPLE 13 In vitro Model for Evaluating Morphogen Species TransportAcross the Blood-Brain Barrier

[0153] Described below is an in vitro method for evaluating the facilitywith which selected morphogen species likely will pass across theblood-brain barrier. A detailed description of the model and protocolare provided by Audus, et al., Ann. N.Y. Acad. Sci 507: 9-18 (1987), thedisclosure of which is incorporated herein by reference.

[0154] Briefly, microvessel endothelial cells are isolated from thecerebral gray matter of fresh bovine brains. Brains are obtained from alocal slaughter house and transported to the laboratory in ice coldminimum essential medium (MEM) with antibiotics. Under sterileconditions the large surface blood vessels and meninges are removedusing standard dissection procedures. The cortical gray matter isremoved by aspiration, then minced into cubes of about 1 mm. The mincedgray matter then is incubated with 0.5% dispase (BMB, Indianapolis,Ind.) for 3 hours at 37° C. in a shaking water bath. Following the 3hour digestion, the mixture is concentrated by centrifugation (1000×gfor 10 min.), then resuspended in 13% dextran and centrifuged for 10min. at 5800×g. Supernatant fat, cell debris and myelin are discardedand the crude microvessel pellet resuspended in 1 mg/mlcollagenase/dispase and incubated in a shaking water bath for 5 hours at37° C. After the 5-hour digestion, the microvessel suspension is appliedto a pre-established 50% Percoll gradient and centrifuged for 10 min at1000×g. The band containing purified endothelial cells (second band fromthe top of the gradient) is removed and washed two times with culturemedium (e.g., 50% MEM/50% F-12 nutrient mix). The cells are frozen (−80°C.) in medium containing 20% DMSO and 10% horse serum for later use.

[0155] After isolation, approximately 5×10⁵ cells/cm² are plated onculture dishes or 5-12 mm pore size polycarbonate filters that arecoated with rat collagen and fibronectin. 10-12 days after seeding thecells, cell monolayers are inspected for confluency by microscopy.

[0156] Characterization of the morphological, histochemical andbiochemical properties of these cells has shown that these cells possessmany of the salient features of the blood-brain barrier. These featuresinclude: tight intercellular junctions, lack of membrane fenestrations,low levels of pinocytotic activity, and the presence of gamma-glutamyltranspeptidase, alkaline phosphatase, and Factor VIII antigenactivities.

[0157] The cultured cells can be used in a wide variety of experimentswhere a model for polarized binding or transport is required. By platingthe cells in multi-well plates, receptor and non-receptor binding ofboth large and small molecules can be conducted. In order to conducttransendothelial cell flux measurements, the cells are grown on porouspolycarbonate membrane filters (e.g., from Nucleopore, Pleasanton,Calif.). Large pore size filters (5-12 mm) are used to avoid thepossibility of the filter becoming the rate-limiting barrier tomolecular flux. The use of these large-pore filters does not permit cellgrowth under the filter and allows visual inspection of the cellmonolayer.

[0158] Once the cells reach confluency, they are placed in aside-by-side diffusion cell apparatus (e.g., from Crown Glass,Sommerville, N.J.). For flux measurements, the donor chamber of thediffusion cell is pulsed with a test substance, then at various timesfollowing the pulse, an aliquot is removed from the receiver chamber foranalysis. Radioactive or fluorescently-labelled substances permitreliable quantitation of molecular flux. Monolayer integrity issimultaneously measured by the addition of a non-transportable testsubstance such as sucrose or inulin and replicates of at least 4determinations are measured in order to ensure statistical significance.

EXAMPLE 14 Screening Assay for Candidate Compounds which AlterEndogenous Morphogen Levels

[0159] Candidate compound(s) which may be administered to affect thelevel of a given morphogen may be found using the following screeningassay, in which the level of morphogen production by a cell type whichproduces measurable levels of the morphogen is determined with andwithout incubating the cell in culture with the compound, in order toassess the effects of the compound on the cell. This can be accomplishedby detection of the morphogen either at the protein or RNA level. A moredetailed description also may be found in U.S. Ser. No. 752,861,incorporated hereinabove by reference.

14.1 Growth of Cells in Culture

[0160] Cell cultures of kidney, adrenals, urinary bladder, brain, orother organs, may be prepared as described widely in the literature. Forexample, kidneys may be explanted from neonatal or new born or young oradult rodents (mouse or rat) and used in organ culture as whole orsliced (1-4 mm) tissues. Primary tissue cultures and established celllines, also derived from kidney, adrenals, urinary, bladder, brain,mammary, or other tissues may be established in multiwell plates (6 wellor 24 well) according to conventional cell culture techniques, and arecultured in the absence or presence of serum for a period of time (1-7days). Cells may be cultured, for example, in Dulbecco's Modified Eaglemedium (Gibco, Long Island, N.Y.) containing serum (e.g., fetal calfserum at 1%-10%, Gibco) or in serum-deprived medium, as desired, or indefined medium (e.g., containing insulin, transferrin, glucose, albumin,or other growth factors).

[0161] Samples for testing the level of morphogen production includesculture supernatants or cell lysates, collected periodically andevaluated for OP-1 production by immunoblot analysis (Sambrook et al.,eds., 1989, Molecular Cloning, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.), or a portion of the cell culture itself, collectedperiodically and used to prepare polyA+ RNA for RNA analysis. To monitorde novo OP-1 synthesis, some cultures are labeled according toconventional procedures with an ³⁵S-methionine/³⁵S-cysteine mixture for6-24 hours and then evaluated to OP-1 synthesis by conventionalimmunoprecipitation methods.

14.2 Determination of Level of Morphogenic Protein

[0162] In order to quantitate the production of a morphogenic protein bya cell type, an immunoassay may be performed to detect the morphogenusing a polyclonal or monoclonal antibody specific for that protein. Forexample, OP1 may be detected using a polyclonal antibody specific forOP-1 in an ELISA, as follows.

[0163] 1 mg/100 ml of affinity-purified polyclonal rabbit IgG specificfor OP1 is added to each well of a 96-well plate and incubated at 37° C.for an hour. The wells are washed four times with 0.167M sodium boratebuffer with 0.15 M NaCl (BSB), pH 8.2, containing 0.1% Tween 20. Tominimize non-specific binding, the wells are blocked by fillingcompletely with 1% bovine serum albumin (BSA) in BSB and incubating for1 hour at 37° C. The wells are then washed four times with BSBcontaining 0.1% Tween 20. A 100 ml aliquot of an appropriate dilution ofeach of the test samples of cell culture supernatant is added to eachwell in triplicate and incubated at 37° C. for 30 min. After incubation,100 ml biotinylated rabbit anti-OP1 serum (stock solution is about 1mg/ml and diluted 1:400 in BSB containing 1% BSA before use) is added toeach well and incubated at 37° C. for 30 min. The wells are then washedfour times with BSB containing 0.1% Tween 20. 100 mlstrepavidin-alkaline (Southern Biotechnology Associates, Inc.Birmingham, Ala., diluted 1:2000 in BSB containing 0.1% Tween 20 beforeuse) is added to each well and incubated at 37° C. for 30 min. Theplates are washed four times with 0.5M Tris buffered Saline (TBS), pH7.2. 50 ml substrate (ELISA Amplification System Kit, Life Technologies,Inc., Bethesda, Md.) is added to each well incubated at room temperaturefor 15 min. Then, 50 ml amplifier (from the same amplification systemkit) is added and incubated for another 15 min at room temperature. Thereaction is stopped by the addition of 50 ml 0.3 M sulfuric acid. The ODat 490 nm of the solution in each well is recorded. To quantitate OP1 inculture media, a OP1 standard curve is performed in parallel with thetest samples.

[0164] Polyclonal antibody may be prepared as follows. Each rabbit isgiven a primary immunization of 100 μg/500 ml E. coli produced OP-1monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1% SDS mixed with 500μl E. coli produced OP-1 monomer (amino acids 328-431 in SEQ ID NO:5) in0.1% SDS mixed with 500 μl Complete Freund's Adjuvant. The antigen isinjected subcutaneously at multiple sites on the back and flanks of theanimal. The rabbit is boosted after a month in the same manner usingincomplete Freund's Adjuvant. Test bleeds are taken from the ear veinseven days later. Two additional boosts and test bleeds are performed atmonthly intervals until antibody against OP1 is detected in the serumusing an ELISA assay. Then, the rabbit is boosted monthly with 100 mg ofantigen and bled (15 ml per bleed) at days seven and ten after boosting.

[0165] Monoclonal antibody specific for a given morphogen may beprepared as follows. A mouse is given two injections of E. coli producedOP1 monomer. The first injection contains 100 mg of OP1 in completeFreund's adjuvant and is given subcutaneously. The second injectioncontains 50 mg of OP-1 in incomplete adjuvant and is givenintraperitoneally. The mouse then receives a total of 230 mg of OP-1(amino acids 307-431 in SEQ ID NO:5) in four intraperitoneal injectionsat various times over an eight month period. One week prior to fusion,both mice are boosted intraperitoneally with 100 mg of OP-1 (307-431)and 30 mg of the N-terminal peptide (Ser₂₉₃-Asn₃₀₉-Cys) conjugatedthrough the added cysteine to bovine serum albumin with SMCCcrosslinking agent. This boost was repeated five days (IP), four days(IP), three days (IP) and one day (IV) prior to fusion. The mouse spleencells are then fused to myeloma (e.g., 653) cells at a ratio of 1:1using PEG 1500 (Boeringer Mannheim), and the cell fusion is plated andscreened for OP1-specific antibodies using OP-1 (307-431) as antigen.The cell fusion and monoclonal screening then are according to standardprocedures well described in standard texts widely available in the art.

EXAMPLE 15 Morphogen-induced Dendritic Growth in Spinal Motor Neurons InVitro

[0166] In order to evaluate the effects of various neurotrophic proteinson neurite outgrowth, dissociated motor neurons from the spinal cordwere exposed OP-1, BDNF, LIF, or GDNF in vitro.

[0167] Suspensions of motor neurons dissociated from the spinal cord ofrat fetuses (E14 day) were prepared and plated essentially according tothe method of Higgins, et al., CULTURING NERVE CELLS, Banker and Goslin,eds., MIT Press, pp. 177-205 (1991), incorporated by reference herein.Neurons were plated at low density (about 15 cells/mm²) ontopoly-D-lysine coated coverslips and maintained in a serum-free medium,Higgins, et al., CULTURING NERVE CELLS (1991). Cytosine-b-D-furanoside(1 μM) was added to the medium of all cultures for 48 hrs on the secondday. This exposure was sufficient to render the cultures virtually freeof nonneuronal cells for 30 days. Exposure to vehicle, OP-1, BDNF, LIF,or GDNF was initiated after the elimination of nonneuronal cells.

[0168] Cellular morphology was routinely assessed by intracellularinjection of fluorescent dyes (4% Lucifer Yellow or 5% 5,6dicarboxyfluorescein; Bruckenstein and Higgins, Dev. Biol. 128: 924-936(1988). Only neurons whose cell bodies were at least 150 mm from theirnearest neighbor were injected, because density-dependent changes inmorphology occur when somata of motor neurons are separated by lesserdistances. Highly purified recombinant human OP-1 was isolated frommedium conditioned by transfected Chinese hamster ovary cells usingS-Sepharose and phenyl-Sepharose chromatography followed by reversephase high performance liquid chromatography as described previously.See Sampath, et al., J. Biol. Chem. 267: 20352-20362 (1992).

[0169] Cultures were immunostained with antibodies previously shown toreact selectively with either axons or dendrites. Lein and Higgins, Dev.Biol. 136: 330-345 (1989). Dendritic probes included mAb to MAP2, tononphosphorylated forms of the M and H neurofilaments, and to thetransferrin receptor. Axonal probes included monoclonal antibodiesagainst to synaptophysin, tau, and phosphorylated forms of the H and theM and H neurofilament subunits. All antigens were localized by indirectimmunofluorescence using previously described procedures. Lein andHiggins, Dev. Biol. 136: 330-345 (1989). Image 1 Software (UniversalImaging) was used for the morphometric analyses of dendritic growth inimmunostained cultures.

[0170] Addition of OP1 enhanced dendritic growth of spinal motorneurons. The dendrites that formed in the presence of OP-1 weresignificantly longer, had a larger number of branchpoints, and had alarger diameter than control spinal motor neurons. The effects of OP-1appeared to be specific to dendrites OP-1 did not significantly affectthe total length of axons. However, axon length was significantlyincreased by LIF and GDNF. These observations are summarized in Table I.TABLE I COMPARISON OF THE EFFECTS OF VARIOUS GROWTH HORMONES ONDENDRITIC GROWTH OF SPINAL MOTOR NEURONS # of Condi- Total dendriticdendritic Least somal Total axonal tion length branchpoints diameterlength Control 65.09 ± 6.7  0.65 ± 0.17  8.81 ± 0.32 180.95 ± 14.5  OP-1133.38 ± 10.0* 1.40 ± 0.22* 10.10 ± 0.44* 200.20 ± 11.9  BDNF 56.58 ±8.6  0.45 ± 0.78  9.56 ± 0.32 158.10 ± 9.8  LIF 59.64 ± 8.3  0.58 ±0.20  8.87 ± 0.29 272.00 ± 24.2* GDNF 52.06 ± 12.6 0.76 ± 0.39  8.66 ±0.24 288.89 ± 23.5*

EXAMPLE 16 Morphogen-Induced Dendritic Growth in Various Neurons InVitro

[0171] In order to further evaluate the effects of morphogens on neuriteoutgrowth, various neuronal populations were exposed OP-1 in vitro.

16.1 Cortical Neurons

[0172] The effects of morphogens on neurite outgrowth were evaluated incortical neurons. Pregnant Balb/c mice (E 18) were euthanised bydecapitation following CO₂ anesthesia and the embryos removed understerile conditions. After carefully removing the meninges, the frontalcerebral cortex was dissected in sterile Hank's balanced salt solution(HBSS) without Ca⁺²/Mg⁺² (Biowhittaker) containing 0.6% glucose and 0.5%HEPES (Sigma). The cortex was minced to 1 mm thick pieces anddissociated into a single-cell suspension using the following protocol.Pieces of frontal cortex were placed in 4.5 ml of Ca⁺²/Mg⁺²-free HBSS ina 50 ml conical culture tube and incubated in a water bath for 5 minutesat 37° C. Then, 0.5 ml of 2.5% trypsin solution (Gibco) was added andthe tissue was then incubated for 10 minutes on a shaking device at 37°C. The supernatant was then removed and placed into another tubecontaining 0.5 ml fetal bovine serum (FBS; Gibco). Five ml of 0.025%Deoxyribonuclease I (Dnase; Calbiochem Corp.) in Ca⁺²/Mg⁺²-free HBSS wasthen added to the pellet and the incubation was continued for another 5min on a shaking device at 37° C. At the end of incubation, the trypsinwas inactivated by adding 0.5 ml FBS. The supernatant collected earlierwas combined with the tissue and the cells were then concentrated bycentrifugation (1000 rpm, 5 min.) and the supernatant was decanted.Fresh medium (2 ml) was added to resuspend the pellet which was furtherdissociated into a single-cell suspension by trituration using apipet-tip.

[0173] The cells were plated in Neurobasal Medium (GIBCO), andsupplemented with B-27 Supplement (GIBCO), 1 mM glutamine andpenicillin/streptomycin. For all experiments, the cortical neurons wereplated at low density (1×10⁴ cells/0.5 ml) on poly-D-lysine (50-100μg/ml; Sigma) coated coverslips inserted into 24-well culture plate(Falcon). Cells were grown for two days in vitro at 37° C. in anatmosphere of 5% CO₂. OP-1 (1, 10, 30, or 100 ng/ml) was added eitherthree hours or 24 hours after plating the cortical neurons. BSA wasadded to all wells at a final concentration of 500 μg/ml prior to addingOP-1. Control cultures consisted of culture medium and medium with BSA500 μg/ml.

[0174] Mouse neurites were immunostained with M6, a mouseneuron-specific monoclonal antibody. Cells were first incubated in 0.1 Mphosphate buffered saline (PBS) containing 1% BSA for 30 min at 25° C.and then exposed to M6 in PBS (1:10) for 24 hrs at 4° C.Immunofluorescent labeling for M6 was carried out using biotinylatedsecondary antibodies anti-rat IgG (Sigma; 3 μg/ml, 1:200, 1 hr at 37°C.) followed by avidin-TRITC conjugate (Sigma; 6.5 μg/ml, 1:400, 1 hr at37° C., in the dark).

[0175] Axons were identified with a rabbit polyclonal antiserum to the200 kDa neurofilament protein (NF-H; Sigma; 1:100, 91 μg/ml). Amonoclonal antibody to microtubule-associated protein 2 (MAP2;Boehringer-Mannheim; 1:100, 20 μg/ml) was used as a specific marker ofdendrites. To visualize these intracellular antigens, cells werepermeabilized with 0.5% triton X-100 (TX) in 0.1 M PBS containing 1% BSAand 4% goat serum (GS; Sigma) for 1 hr at 25° C. Primary antibodies werediluted in 0.1M PBS, 1% BSA, 4% GS with 0.5% TX and incubated for 1 hrat 37° C. After the primary incubation, the cells were washed threetimes in PBS containing 4% GS. Labeling was detected with fluorescein-or rhodamine-conjugated antibodies (1:400 in PBS, BSA, GS, and TX, 1 hrat 37° C., in the dark). Mouse antibodies were visualized withfluorescein-coupled goat anti-mouse Ig (Boehringer-Mannheim). Rabbit orrat antibodies were visualized using indirect immunofluorescence withrhodamine-conjugated goat anti-rat or anti-rabbit Ig. Cultures wereadditionally stained with a nuclear stain, 4′,6-Diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma; 0.1μg/ml, 5 min at room temperature). Coverslips were washed once insterile water and let dry for 10 minutes before mounting onto glassslides in aqueous mounting solution (Fluoromount; SouthernBiotechnology). Slides were kept refrigerated in the dark untilexamined.

[0176] Immunoreactive cells were examined in six different microscopicfields selected at random on a minimum of five coverslips for eachexperiment. Three experiments for each condition were carried out. Onlyisolated neurons whose cell bodies or processes that were not in contactwith other neurons were analyzed. A total of 100 neurons were examinedfor each experimental condition.

[0177] For measurements of neurite length, neurons were examined at afinal image magnification of 400×. Fluorescent images of the neuronswere recorded with a CCD video camera (Dage) and analyzed with aMacintosh PowerMac (9500/200) and image processing program (NIH Image1.59). Neurite lengths were measured by tracing the total length of anyneurite extending from a neuron cell body. Recorded lengths werecalibrated at the same magnification using a ciroscope slide micrometer.The number of primary dendrites per cell, the length of major neuritesor axons, the length of primary dendrites, the length and number ofsecondary dendrites, the total length of primary or secondary dendrites,and the total neurite and dendrite length per cell were calculated.Analysis of statistical significance of any observed differences betweenmonolayers was performed using Student's t-test or ANOVA (SPSS/Mac,version 6.1, SPSS Inc., Chicago, Ill.).

[0178] OP-1 enhanced dendritic growth of cortical neurons. The length ofprimary dendrites and the number and length of secondary dendrites weresignificantly increased in the presence of OP-1. The increase indendritic growth was dose-dependent. Maximal growth was observed in thepresence of 30-100 μg/ml of OP-1. The effects of OP-1 appeared to bedendrite-specific; OP1 did not significantly affect the cell morphology,body size, and axon length of cortical neurons. These observations aresummarized in Table II. TABLE II EFFECTS OF OP-1 ON DENDRITIC GROWTH OFCORTICAL NEURONS Cell Body Condition Diameter Axon Length # Primary #Secondary BSA 3 hrs 15.9 ± 0.5 153.2 ± 9.1 3.5 ± 0.2  0.4 ± 0.14 OP-1, 1μg, 3 hrs 15.98 ± 0.5  151.1 ± 6.2 3.5 ± 0.2 0.55 ± 0.13 OP-1, 10 μg, 3hrs  17.8 ± 0.49 139.3 ± 8.5  4.0 ± 0.14  0.6 ± 0.13 OP-1, 30 μg, 3 hrs17.74 ± 0.5  134.5 ± 6.9  4.0 ± 0.15 0.96 ± 0.14 OP-1, 100 μg, 3 hrs17.3 ± 0.4 138.8 ± 6.6 3.6 ± 0.2 1.3 ± 0.2 BSA 24 hrs  16.3 ± 0.45 144.8± 5.9  3.5 ± 0.17 0.48 ± 0.11 OP-1, 1 μg, 24 hrs 15.6 ± 0.5  142.6 ±10.9 3.4 ± 0.2 0.4 ± 0.1 OP-1, 10 μg, 24 hrs 16.7 ± 0.4  144.2 ± 8.16 3.2 ± 0.14 0.6 ± 0.1 OP-1, 30 μg, 24 hrs 16.5 ± 0.4 139.4 ± 7.8 3.5 ±0.2 0.86 ± 0.12 OP-1, 100 μg, 24 hrs 17.3 ± 0.4  156.5 ± 11.1  3.0 ±0.19 0.86 ± 0.17 Length Length Total Total Total Condition PrimarySecondary Primary Secondary Dendrite BSA 3 hrs 15.4 ± 0.6 3.4 ± 0.8 51.4± 4.5 3.6 ± 1.5 54.9 ± 5.1 OP-1, 1 μg, 3 hrs 14.2 ± 0.4 4.7 ± 0.7 49.1 ±3.5 5.7 ± 1.5 54.8 ± 4.6 OP-1, 10 μg, 3 hrs 20.5 ± 0.6 4.6 ± 0.6 81.6 ±3.5 5.8 ± 1.2 87.5 ± 4.3 OP-1, 30 μg, 3 hrs 22.9 ± 0.5 7.6 ± 0.7 89.9 ±4.6 11.1 ± 1.9  99.6 ± 5.7 OP-1, 100 μg, 3 hrs 23.7 ± 0.6  9.9 ± 0.7587.7 ± 5.3 16.2 ± 2.9  103.9 ± 7.6  BSA 24 hrs 16.7 ± 0.5 4.6 ± 0.6 55.9± 3.5 4.7 ± 1.1 60.6 ± 4.1 OP-1, 1 μg, 24 hrs 13.7 ± 0.5 3.7 ± 0.7 46.3± 3.6 4.1 ± 1.0 50.4 ± 4.0 OP-1, 10 μg, 24 hrs 16.3 ± 0.6 4.8 ± 0.7 50.8± 3.1 5.3 ± 7.7 56.1 ± 3.6 OP-1, 30 μg, 24 hrs 18.9 ± 0.5 7.4 ± 0.8 65.6± 4.5 9.6 ± 1.6 75.5 ± 5.1 OP-1, 100 μg, 24 hrs 22.1 ± 0.8 7.6 ± 1.064.0 ± 6.4 10.5 ± 2.2  74.8 ± 7.8

16.2 Hippocampal Neurons

[0179] The effects of morphogens on neurite outgrowth were evaluated inhippocampal neuerons. Primary hippocampal cultures were preparedaccording to the method of Banker, et al. See Banker & Cowan Brain Res.126: 397-425 (1977); Banker & Goslin, CULTURING NERVE CELLS (1991). Avery low density of neurons was plated over a monolayer of glial cellsplated on poly-D-lysine coated coverslips and maintained in a serum-freemedium. Cellular morphology was assessed by immunostaining for MAP2.Dendritic length and branching was quantified using the Shoil concentricring analysis. Under these control conditions, hippocampal neuronsproduce 4-6 minor processes. Over the first 24-48 hours, one of theprocesses grows rapidly and becomes an axon. The other processes extendvery slowly and develop into mature dendrites after 6-10 days.

[0180] Because glial cells secrete trophic factors into the medium thatare critical for the development of hippocampal neurons, this culturemethod was modified to assess the effects of OP-1. Hippocampal neuronswere plated in a serum-free medium without glial cells. In the absenceof glial cells, OP-1 markedly enhanced the rate and extent of dendriticdevelopment of hippocampal neurons cultured in serum-free medium.OP-1-treated neurons had significantly increased number of Shoil ringintersections (39.6 vs. 16.02), dendritic length (FIGS. 6 and 7) andnumber of terminal branches (13.05 vs. 6.64; FIG. 8). There were nosignificant differences in the number of primary dendrites. The effectsof OP-1 appeared to be dendrite-specific in this cell type. Asillustrated in FIG. 6; OP-1 did not significantly affect the totallength of axons.

16.3 Sympathetic Neurons (i) Effects of OP-1 on Dendritic Growth

[0181] In order to assess the effects of morphogens on sympatheticneurons, suspensions of neurons dissociated from the superior cervicalganglia of Sprague-Dawley rat fetuses (19-21 day) or rat pups (1-3 daypostnatal) were exposed to OP-1. The suspension were prepared accordingto the method of Higgins, et al., CULTURING NERVE CELLS, Banker andGoslin, eds., MIT Press, pp. 177-205 (1991), the teachings of whichherein incorporated by reference. Equivalent results were obtained withpre- and postnatal animals. Neurons were plated at low density (about 15cells/mm 2) onto poly-D-lysine coated coverslips and maintained in aserum-free medium (Higgins, et al., CULTURING NERVE CELLS (1991))containing NGF (100 ng/ml). Cytosine-b-D-furanoside (1 μM) was added tothe medium of all cultures for 48 hrs on the second day. This exposurewas sufficient to render the cultures virtually free of nonneuronalcells for 30 days. To label sympathetic neuroblasts, ganglia from 15-dayembryos were grown in explant culture for 18 hrs in the presence of³H-(methyl)-thymidine (0.3 mCi/ml, ICN) before being dissociated.Because NT3 (50 ng/ml) enhances the survival of immature sympatheticneurons, Birren, et al., Develop. 119: 597-610 (1993), it was added tothe NGF-containing medium during the period of explant culture and thenext 4 days in vitro. As in cultures of sympathetic neurons, exposure toNGF, OP-1, or both, was initiated after the elimination of nonneuronalcells.

[0182] Cellular morphology was routinely assessed by intracellularinjection of fluorescent dyes (4% Lucifer Yellow or 5% 5,6dicarboxyfluorescein; Bruckenstein and Higgins, Dev. Biol. 128: 924-936(1988)). Only neurons whose cell bodies were at least 150 mm from theirnearest neighbor were injected, because density-dependent changes inmorphology occur when somata of sympathetic neurons are separated bylesser distances. Highly purified recombinant human OP-1 was isolatedfrom medium conditioned by transfected Chinese hamster ovary cells usingS-Sepharose and phenyl-Sepharose chromatography followed by reversephase high performance liquid chromatography as described previously.See Sampath, et al., J. Biol. Chem. 267: 20352-20362 (1992).

[0183] Under control conditions, sympathetic neurons typically extendeda single process during the first 24-48 hrs in vitro. This process hasthe cytoskeletal and ultrastructural characteristics of an axon. Theaxon continued to elongate during the next few weeks and generate anelaborate plexus. The basic morphology of the cells, however, remainedessentially unchanged, with 80% of the neurons still being unipolarafter one month in vitro. Most of the remainder had either two axons(13% of the cells) or an axon and a short dendrite (7%). Thus, the meannumber of processes at this time was 1.13±0.06 axons/cells and 0.07±0.04dendrites/cell.

[0184] Exposure to OP-1 caused sympathetic neurons to form additionalprocesses. This response was relatively slow, with only 42% of the cellsforming a second process within 24 hrs. However, virtually all cells(94%) had begun to respond to maximally effective concentrations of OP1within three days. The processes that formed in the presence of OP-1 hadthe appearance of dendrites. These processes were broad-based (up to 5μm diameter), exhibited a distinct taper, and branched in a “Y”-shapedpattern, with daughter processes being distinctly smaller than theparent process. Dendrites were much thicker than sympathetic axons and,unlike axons, they ended locally, usually extending less than 300 mmfrom the soma. The mean number of dendrites/cell continued to increaseduring a four week exposure to OP-1, with most of the change occurringduring the first 10 days of treatment. After four weeks, OP-1-treatedneurons had a mean of 7.3±0.3 dendrites/cell, representing a 100-foldincrease over control cells. During this time, the size of the dendriticarbor also increased, with cells progressing from simple cells to a morecomplicated morphology. These observations are summarized in panels A, Band C of FIG. 9.

[0185] The effects of OP-1 appeared to be dendrite-specific in this celltype. The effects of OP-1 on initial axon growth during the first 48 hrsin culture were examined. OP-1 did not affect the rate at which axonswere initially extended, or the number of axons extended per cell. Cellnumber also remained constant during the exposure to OP-1, indicatingthat the morphogen was not acting by enhancing the survival of asubpopulation of neurons, as shown in panel C of FIG. 9. The effects ofOP-1 were also examined in the delayed introduction paradigm, and noincrease in axon number was observed.

(ii) Effects of Various Concentrations of OP-1 on Dendritic Growth

[0186] In order to assess whether the effects of morphogens on dendriticgrowth were concentration-dependent, suspensions of neurons dissociatedfrom the superior cervical ganglia were exposed to variousconcentrations of OP-1.

[0187] The effects of OP1 were concentration-dependent (FIG. 10).Significant changes in dendritic growth could be detected withconcentrations as low as 300 pg/ml, and half-maximal effects wereobserved at about 2 ng/ml. Maximal dendritic growth was obtained withmedial concentrations between 30 and 100 ng/ml. Although typically addedto the medium on day 5, earlier initiation of dendritic growth (by thethird day of culture) could be obtained by adding OP1 to the medium atthe time of plating. No dendritic growth was detected in cultures inwhich the OP1 (1 μg/ml) had been allowed to absorb to coverslips beforeplating cells.

[0188] It appeared that several parameters of dendritic growth,including the percentage of cells with dendrites, the mean number ofdendrites/cell, dendritic length (not shown), changed over the sameconcentration range. In addition, three other changes were observed incellular morphology in this morphogen concentration range. As had beenobserved while dendritic growth is occurring in vivo, the somata becamelarger. In addition, the nuclei became less eccentric, and the axonsformed small fascicles.

(iii) Effects of Various Morphogens on Dendritic Growth

[0189] Using the methods described above, other morphogens were testedfor their capacity to induce dendritic growth of sympathetic neurons andtheir effects on the expression of cytoskeletal proteins. Sympatheticneurons were dissociated from the superior cervical ganglia of Holtzman(Harlan Sprague-Dawley) rat fetuses (E21) or pups (1 day postnatal) andplated onto poly-D-lysine-coated coverslips according to the method ofHiggins, et al., CULTURING NERVE CELLS, Banker and Goslin, eds., MITPress, pp. 177-205 (1991). The cells were maintained in a serum-freemedium that contains nerve growth factor (100 ng/ml), and nonneuronalcells were eliminated by exposure to cytosine-β-D-arabinofuranoside (1μM) for 48-72 h beginning on the second day after plating. Themorphology of the neurons was routinely assessed by intracellularinjection of the fluorescent dye Lucifer yellow (4%) and byimmunostaining with dendrite-specific antibodies. These includedmonoclonal antibody SMI 32 (Stemberger Monocionals, Inc.) whichrecognizes nonphosphorylated epitopes on the H and M neurofilamentsubunits and AP20 (Sigma) and SMI 52 (Stemberger Monocionals, Inc.)which both react with microtubule-associated protein-2 (MAP2). Highlypurified recombinant human proteins (BMP-2, BMP-3 and CDMP-2) andDrosophila 60A were prepared as described previously for OP-1.

[0190] For Western blot analysis of cytoskeletal proteins, sympatheticneurons were plated onto poly-D-lysine coated 35 mm dishes and treatedwith 50 ng/ml of BMP-2, OP-1, 60A or CDMP-2 for five days. Cells werethen scraped off dishes in 50 mM Tris buffer (pH 7.4) containing 0.1%SDS, 2% 2-mecaptoenthanol and 1 mM EDTA and homogenized by passingthrough a 23 gauge needle at 4° C. Cell extracts were centrifuged at12000×g for 15 minutes and the protein concentrations of thesupernatants were determined using the Bradford dye reagent (Bio-Rad).Equal amounts of proteins were resolved by SDS-PAGE, electrophoreticallytransferred onto a nitrocellulose membrane, and probed with antibodiesto MAP2 or an antibody SMI 31 (Stemberger Monocionals, Inc.) to thephosphorylated forms of the H and M neurofilament subunits. Detectionwas accomplished using Chemiluminescent Substrate (Pierce Chemical Co.)after sequential treatment with biotinylated goat antimouse IgG (HyCloneLaboratories, Inc.) and with horseradish peroxidase-conjugatedstreptavidin (Amersham).

[0191] As illustrated in FIG. 11, all the morphogens tested (i.e., OP-1,BMP-2, BMP-3, CDMP-2, and 60A) induced significant dendritic growth insympathetic neurons. However, significant variations in efficacy wereobserved. Treatment with maximally effective concentrations (50 ng/ml)of BMP-2 or OP-1 for five days caused virtually all of the neurons toform dendrites (Table III). These processes exhibited a distinct taper,branched at “Y” shaped angles, and extended approximately 100 μm fromthe cell bodies after five days of treatment. Examination ofconcentration effect relationships (FIG. 11) revealed that the EC₅₀ forBMP-2 (1.7 ng/ml) was similar to that for OP-1 (1.8 ng/ml) and thatmaximally effective concentrations of these two growth factors hadequivalent effects on the cells, as assessed by both the number ofdendrites per cell and the length of the longest dendrite (Table III).Moreover, the effects of OP-1 and BMP-2 were not additive (data notshown) suggesting that the two ligands may share aspects of a commonsignaling pathway. The Drosophila 60A protein also stimulated dendriticgrowth and the EC₅₀ (2.7 ng/ml) for this activity was similar to thatfor OP-1 and BMP-2. However, 60A was less efficacious, and at maximallyeffective concentrations caused cells to form fewer dendrites (1.2/cell)than either OP-1 or BMP-2 (4.6 or 4.9/cell, respectively). BMP-3 andCDMP-2 only produced a statistically significant increase in dendriticgrowth at the highest concentration tested (100 ng/ml). The differentefficacies for promoting dendrite growth may indicate relativelystringent structural requirements for this biological activity. OP-1,BMP-2 and 60A, which share a high sequence homology (89-90%) in theconserved seven-cysteine skeleton sequence, had much higher efficaciesthan BMP-3 and CDMP-2, which share 78% and 82% homology, respectively,with the reference sequence of OP-1. See FIG. 1. TABLE III COMPARISON OFTHE EFFECTS OF VARIOUS MORPHOGENS ON DENDRITIC GROWTH OF SYMPATHETICNEURONS Growth Dendrites % Cells with Length of the EC₅₀ Factor per CellDendrites Longest Dendrite (pm) (ng/ml) Control 0.20 ± 0.14  13.3  6.0 ±4.3 OP-1 4.62 ± 0.49* 100.0  112.3 ± 5.6  1.84 BMP-2 4.93 ± 0.46* 100.0 107.1 ± 5.6  1.66 60A 1.20 ± 0.26* 73.3 50.7 ± 9.0 2.70 BMP-3 0.44 ±0.22  25.0 16.3 ± 7.6 26.05  CDMP-2 0.25 ± 0.14  18.8 12.5 ± 6.7 98.38 

[0192] The effects of various morphogens on the expression ofcytoskeletal proteins were also assessed using methods described above.After nonneuronal cells had been eliminated, sympathetic neurons weretreated with control medium or with 50 ng/ml of OP-1, BMP-2, 60A orCDMP-2, for five days. Cultures were then solubilized and subjected toWestern blot analysis for MAP2 (primarily located in dendrites) and forphosphorylated forms of the H and M neurofilament subunits (primarilylocated in axons). The efficacy of the various morphogens in increasingMAP2 expression correlated with their ability to induce dendriticgrowth. Cultures exposed to BMP-2, OP-1 or 60A exhibited significantincreases (3.0±0.4, 2.3±0.4, and 1.8±0.3 fold, respectively) in theexpression of high molecular weight forms of MAP2 when compared tocontrol cultures. The level of expression of MAP2 was not significantlyincreased in cultures exposed to CDMP-2. None of the morphogens testedaffected the expression of the phosphorylated forms of the H and Mneurofilament subunits. These results show that morphogens enhance theexpression of a microtubule-associated protein which is found indendrites and which is required for the growth of these processes. Theseobservations suggest that regulation of MAP2 expression may be one ofthe mechanisms by which morphogens regulate the morphologicaldevelopment of sympathetic neurons.

(iv) Comparison of OP-1 to Other Growth Factors

[0193] In order to evaluate whether the effects on dendritic outgrowthare specific to morphogens, the effects of other growth factors ondendritic growth were compared to those of OP-1.

[0194] Mature human recombinant OP-1 was isolated from mediumconditioned by transfected Chinese hamster ovary cells using S-Sepharoseand phenyl-Sepharose chromatography followed by reverse phase highperformance liquid chromatography as described above. Ciliaryneurotrophic factor (CNTF) was purified from rat sciatic nerveManthorpe, et al., Brain Res. 367: 282-286 (1986), the teachings ofwhich are herein incorporated by reference) and activin A was generouslyprovided by Ralph Schwall (Genentech). Other growth factors wereobtained from commercial sources: GIBCOBRL (IL-1β, IL-3, IL-4, IL-6,IL-7; LIF, EGF, GM-CSF, RANTES, MCAF, TGF-α, TGF-β1 and 3, rat gammainterferon); Collaborative Research (HGF, PDGF); Boehringer (IL-2); andPromega (IL-8); R&D Systems (BDNF, NT3, NT4, bFGF).

[0195] As summarized in Table IV, dendritic growth was not observed inthe presence of TGF-β1, TGF-β3, activin A or inhibin, all of which aremembers of the TGF-β family but are not members of the structurally andfunctionally distinct morphogen sub-family. In addition, negativeresults were obtained with most neurotrophins and nine other growthfactors known to affect neuronal survival or differentiation (Table IV).In other experiments, negative results were also obtained with: TGF-β2,interleukins 1β, 2, 3, 4, 6, 7, and 8, PDGF, HGF, GM-CSF, MCAF, RANTES,TGF-α and gamma interferon. Thus, it would appear that thedendrite-promoting effect of morphogens is a highly specific responsethat is observed with a very limited subset of growth factors. TABLE IVCOMPARISON OF THE EFFECTS OF OP-1 AND OTHER GROWTH FACTORS ON DENDRITICGROWTH MEAN NUMBER OF GROWTH FACTOR DENDRITES/CELL NONE  0.8 ± 0.04 OP-13.08 ± 0.20 TGF-β1 0.17 ± 0.09 TGF-β3 0.00 ± 0.00 INHIBIN 0.20 ± 0.10ACTIVIN A 0.08 ± 0.05 BDNF 0.11 ± 0.05 NT3 0.11 ± 0.07 NT4 0.32 ± 0.11CNTF 0.10 ± 0.05 LIF 0.13 ± 0.07 EUF 0.07 ± 0.07 bFGF 0.03 ± 0.03

(v) Role of Morphogens in Glial-Induced Dendritic Growth

[0196] Sympathetic neurons extend only a single axon when grown in theabsence of serum or nonneuronal cells. In contrast, co-culturingsympathetic neurons with glial cells causes these neurons to formdendrites. In order to assess the potential role of morphogens inglial-induced dendritic growth, neuron and glial cells were co-culturedin the presence of a monoclonal antibody (mAb) raised against hOP-1.

[0197] Dendritic growth in sympathetic neurons grown with astrocytes orSchwann cells was inhibited by 40-60% in the presence of a hOP-1 mAb.SDS-PAGE analyses by hOP-1 mAb of proteins immunoprecipitated fromneuron-glia co-cultures revealed several bands, the molecular weights ofwhich corresponded to the cellular and secreted forms of hOP-1 .Immunocytochemical analyses of co-cultures indicate that both neuronsand glia express cytoplasmic and surface staining for OP1 and BMP-6.Similar patterns of immunoreactivity were observed in glia grown in theabsence of neurons. However, neurons cultured in the absence of gliaexpressed cytoplasmic but not surface staining for OP-1 or BMP-6. Thesedata are consistent with a role for morphogens in glia-induced dendriticgrowth.

EXAMPLE 17 Morphogen-Induced Synaptic Formation

[0198] As described in Examples 15 and 16, OP-1 induces dendritic growthin various populations of cultured neurons. To determine if thesedendrites are receptive to innervation, OP-1-treated culturedhippocampal neurons were immunostained with antibodies to MAP2 andsynapsin. Sites of presynaptic contact were defined by puncta ofsynapsin immunoreactivity. Given the poor growth of axons in culturedhippocampal neurons maintained in a serum-free medium, a heterochronicculture technique was used to assess the ability of the OP-1-extendeddendrites to receive axonal contacts. Cultured neurons were grown in thepresence of OP1 for three days. New neurons were plated on top of thesemore mature neurons and fixed one day later. Previous work has shownthat axonal contacts will form within 24 hours of plating if more maturedendrites are present within the culture. Fletcher, et al., J. Neurosci14: 6695-6706 (1994). Using this heterochronic culture technique,synapsin positive aggregates were found surrounding OP-1-induceddendrites. As illustrated in FIG. 12, OP-1-reated cultured hippocampalneurons had a significantly higher number of synapses per neuron thanuntreated neurons or neurons co-cultured with glial cells. Theseobservations suggest that the OP1 induced dendritic outgrowth producesdendrites that are receptive to innervation.

EXAMPLE 18 Morphogen-induced Dendritic Growth and Synaptogenesis In Vivo

[0199] In order to assess the effects of morphogen on dendritic growthin vivo, rats are injected intraperitoneally once per day with OP1 atdose of 2 mg/kg. The control group consists of rats injectedintraperitoneally with the vehicle (20 mM arginine (pH 9.0), 150 mM NaClwith 0.1% Tween 80). After seven days, rats are anesthetized with etherand the superior cervical ganglia, hippocampus, and hypoglossal nucleusare removed. Subsequently, rats are perfused with paraformaldehyde andthe kidney and retina are removed.

[0200] Superior cervical ganglia are desheathed and pinned in a chambersuperfused with an oxygenated physiological saline. For intracellularstaining, neurons are impaled with triangular glass electrodes filledwith a 4% solution of horseradish peroxidase (HRP). HRP is introducedinto the cell by iontophoresis and the reaction product is visualized bythe pyrocathecol-phenylenediamine method. Hanker, et al., Histochem. J.9: 789-792 (1977); for details see Purves and Hume, J. Neurosci. 1:441-452 (1981); Forehand and Purves, J. Neurosci. 4: 1-12 (1984). Fiveto ten cells/ganglion are injected. After allowing two hours for dyediffusion, the ganglia are fixed in 4% formaldehyde overnight. Afterdehydration, stained neurons are viewed at 300× in whole-mountpreparations and traced with the aid of a camera lucida.

[0201] To confirm the light microscopic identification of processes andto assess the state of differentiation of the dendrites formed in thepresence of OP-1, superior cervical ganglia are immunostained withantibodies previously shown to react selectively with either axons ordendrites. Lein and Higgins, Dev. Biol. 136: 330-345 (1989). Monoclonalantibodies (mAb) to MAP2 (e.g., AP14), to nonphosphorylated forms of theM and H neurofilaments (SMI 32, Stembery-Meyer Immunocytochemicals), andto the transferrin receptor (MRC OX-26, Serotech) are used as dendriticmarkers and mAb to synaptophysin (SY-38, Boehringer Mannheim), tau(e.g., Tau 1), and phosphorylated forms of the H (NE14, BoehringerMannheim) and the M and H (SMI 31, Stembery-Meyer Immunocytochemicals)neurofilament subunits are used as axonal markers. All antigens arelocalized by indirect immunofluorescence using previously describedprocedures. Lein and Higgins, Dev. Biol. 136: 330-345 (1989). Image 1Software (Universal Imaging) is used for the morphometric analyses ofdendritic growth in immunostained cultures. In addition, in order todetermine the effects of OP1 on synaptogenesis in superior cervicalganglia in vivo, neurons are immunostained with antibodies to synapsin.Sites of presynaptic contact are defined by puncta of synapsinimmunoreactivity.

[0202] Hippocampal or hypoglossal tissue is impregnated with GolgiCoxsolution. Following dehydration, the tissue is embedded in celloidin andsectioned at 160 μm on a microtome. Sections are then developed in 5%sodium sulphite and mounted on a glass slide with permount. Kidney andretinal tissue is removed from animals that have been perfused withformaldehyde. The fixed tissue is embedded in paraffin and sectioned at160 μm on a microtome. Sections are then developed in 5% sodium sulphiteand mounted on a glass slide with permount. Sections of hippocampal,hypoglossal, kidney, and retinal tissue are immunostained withantibodies previously shown to react selectively with axons, dendrites,or synapsin. Antigens are localized by indirect immunofluorescence, asdescribed above.

[0203] Dendritic and axonal processes are distinguished usingestablished criteria. Purves and Hume, J. Neurosci. 1: 441-452 (1981).Dendrites have numerous short processes arising from the main shaft andbranched into secondary and tertiary segments relatively close to thecell soma. The axon is readily identified as a smooth, thick processthat usually could be followed for at least several hundred microns andfrequently can be seen exiting the ganglion via a postganglionic nerve.The arbor of each neuron is assessed by four measures of dendriticcomplexity. The number of primary dendrites is determined by viewing thecells at 480× in multiple focal planes. A primary dendrite is defined asany process extending from the soma a distance greater than one celldiameter. Total dendritic lengths are measured from the camera lucidatracings with the aid of a digitizing tablet and a general purposeprogram for neural imaging. Voyvodic, Soc. Neurosci. Abstr. 12: 390(1986). The radius of a circle incorporating the entire arbor ismeasured as an indicator of the process length. Finally, the extent ofbranching is determined by counting the number of branches crossing a50% circle. Scholl, J. Comp. Neurol. 244: 245-253 (1953). Sites ofpresynaptic contact are defined by puncta of synapsin immunoreactivity.

[0204] Animals treated with OP-1 are expected to have significantlyenhanced dendritic growth when compared to control animals, reflected inincreased length, diameter, and number of processes. Further, animalstreated with OP-1 are expected to have significantly increased number ofsynaptic contact when compared to control animals.

EXAMPLE 19 Intra-ocular Transplants

[0205] Intra-ocular grafting is a well established model which offers anisolated environment in which CNS synaptic contact can be selectivelyactivated and pharmacologically characterized using drug superfusiontechniques in vivo. See, for example, Olson, et al., ADVANCES INCELLULAR NEUROBIOLOGY, (Academic Press, 1983) Grafts of identified CNSareas are placed into the anterior eye chamber of syngeneic andallogeneic host rats. The development and overall structuralorganization of the graft is relatively organotypic in nature and themature transplant usually provides an in vivo replica of the gratedarea. Hoffer, et al., Brain Res. 79: 165-184 (1974). After maturation ofthe transplant, host animals can be anesthetized and pre andpostsynaptic activity can be examined using in vivo electrochemical andelectrophysiological techniques. Eriksdotter-Nilsson et al., Brain Res.478: 269-280 (1989); Eriksdotter-Nilsson, et al., Exp. Brain Res. 74:89-98 (1989); Hoffer, et al., Brain Res. 79: 165-184 (1974); Johansson,et al., Exp. Neurol. 134: 25-34 (1995). After sacrifice of the hostanimal, grafts and underlying irides can be processed for histochemicalevaluations of neural and glial elements, and for localization ofvarious transmitter-specific structures and receptors. Bjorklund, etal., Dev. Brain Res. 6: 130-140 (1983); Bergman, et al., Hippocampus 2:339-348 (1992); Henschen, et al., Prog. Brain Res. 78: 187-191 (1988);Henschen, et al., Neuroscience 26: 193-213 (1988); and Henschen, et al.,Brain Res. 36: 237-247 (1988). Using sequential grafting of fetal braintissue pieces to the anterior chamber of the eye, it is possible tostudy the conditions under which mature brain and spinal cord tissue(grafts which have resided in oculo for one or more months and which nolonger show morphological signs of growth or development) will acceptingrowth of nerve fibers. Olson, et al., Brain Res. Bull. 9: 519-537(1982). Thus, using the in oculo technique, isolated replicas of definedpathways suitable for structural and functional studies of CNSconnectivity can be obtained.

[0206] In particular, it has been previously shown that grafted spinalcord will survive and grow in oculo in a manner suggesting that ispossesses a considerable intrinsic determination of its normaldevelopment. Henschen, et al., Exp. Brain Res. 60: 38-47(1985). Further,it has been previously demonstrated that models of the descendingcoeruleo-spinal noradrenergic and bulbospinal serotonergic pathways tospinal cord can be generated when these CNS areas are co-grafted inoculo. Henschen, et al., Brain Res. Bull. 15: 335-342 (1985); Henschen,et al., Brain Res. Bull. 17: 801-808 (1986); Henschen, et al., Dev.Brain Res. 36: 237-247 (1987); Henschen, et al., Exp. Brain Res. 75:317-326 (1989). In a similar vein, corticospinal and sensory and motorpathways can be constructed in oculo, using co-grafts of cerebral cortex(Palmer, et al., Exp. Brain Res. 87: 96-107 (1991); dorsal root ganglia(Trok, et al., 1997), and muscle (Trok, et al., Brain Res. 659: 138-146(1994), with subsequent histological and electrophysiological analysis.Thus, in oculo grafts provide a unique isolated system of a much more“in vivo” than “in vitro” nature, to study spinal cord connectivity andresponse to injury.

[0207] In oculo transplants of specific CNS areas have been employed toevaluate of various putative neurotrophic effects: NGF in hippocampus(Eriksdotter-Nilsson et al., Brain Res. 478: 269-280 (1989) andEriksdotter-Nilsson, et al., Exp. Brain Res. 74: 89-98 (1989)), FGF incortical areas (Giacobini, et al., Exp. Brain Res. 86: 73-81 (1991)),IGF-1 in olfactory bulb (Giacobini, et al., 1995)), GDNF in midbraindopaminergic nucleus and spinal cord (Johansson, et al., Exp. Neurol.134: 25-34 (1995); Trok, et al., Neuroscience 71: 231-241 (1996); andTrok, et al., (1996)), and BDNF, NT-3, NT-4, and CNTF in spinal cord(Trok, et al., 1997).

20.1 Effects of OP-1 on Intra-ocular Spinal Cord Transplants

[0208] In oculo transplants were employed to evaluate the effects ofmorphogens on motor neurons. Fisher 344 rats were implanted withsyngeneic E18 spinal cord grafts, essentially as previously described byHenschen, et al., Prog. Brain Res. 78: 187-191 (1988). OP-1 (0.5 μg) orvehicle was injected into the anterior chamber at weekly intervals. Eachrat had an OP-1-treated graft in one eye and a control graft in thecontralateral eye. Survival and growth of the graft was followednoninvasively by observation through the cornea over a four week period.After sacrifice of the host animals, grafts were evaluated byhistological and immunocytochemical techniques. Olson, L., et al.,ADVANCES IN CELLULAR NEUROBIOLOGY, pp. 407-442 (Academic Press, 1983);Granholm, et al., Exp. Neurol. 118: 7-17 (1992).

[0209] As shown in FIG. 13, OP-1-treated grafts maintained asignificantly larger size over the four week observation period,compared to control grafts. Transplants treated with weekly injectionsof vehicle had minimal survival but manifested a marked reduction insize, similar to what has been previously described for El 8 donors.Henschen, et al., Prog. Brain Res. 78: 187-191 (1988); Henschen, et al.,Neuroscience 26: 193-213 (1988). In contrast, grafts treated with weeklyinjections of 0.5 μg of OP-1 maintained a much greater size and, at theend of four weeks, had a size equal to, or only slightly less than, theinitial size at grafting.

[0210] This positive effect of OP-1 was confirmed usingimmunocytochemical techniques. Overall neuron density was evaluatedusing MAP2 (i.e., neurofilament immunoreactivity). As seen in FIG. 14,the number of neurofilament-positive neuronal structures wassignificantly higher in OP-1 -treated grafts compared to vehicle-treatedtransplants. In order to assess more specifically the effects of OP1 onmotor neurons, immunocytochemical studies were carried out using cholineacetyltransferase (CHAT) immunocytochemistry. The number of cholinergiccell bodies and fibers was also significantly higher in OP-1-treatedgrafts than in vehicle-treated transplants. See FIG. 15.

EXAMPLE 20 Traumatic Injury Model

[0211] The fluid percussion brain injury model was used to assess theability of morphogens to restore central nervous system functionsfollowing significant traumatic brain injury.

I. Fluid Percussion Brain Injury Procedure

[0212] The animals used in this study were male Sprague-Dawley ratsweighing 250-300 grams (Charles River). The basic surgical preparationfor the fluid-percussion brain injury has been previously described.Dietrich, et al., Acta Neuropathol. 87: 250-258 (1994) incorporated byreference herein. Briefly, rats were anesthetized with 3% halothane, 30%oxygen, and a balance of nitrous oxide. Tracheal intubation wasperformed and rats were placed in a stereotaxic frame. A 4.8-mmcraniotomy was then made overlying the right parietal cortex, 3.8 mmposterior to bregma and 2.5 mm lateral to the midline. An injury tubewas placed over the exposed dura and bonded by adhesive. Dental acrylicwas then poured around the injury tube and the injury tube was thenplugged with a gelfoam sponge. The scalp was sutured closed and theanimal returned to its home case and allowed to recover overnight.

[0213] On the next day, fluid-percussion brain injury was producedessentially as described by Dixon, et al., J. Neurosurg. 67: 110-119(1987) and Clifton, et al., J. Cereb. Blood Flow Metab. 11: 114-121(1991). The fluid percussion device consisted of a saline-filledPlexiglas cylinder that is fitted with a transducer housing and injuryscrew adapted for the rat's skull. The metal screw was firmly connectedto the plastic injury tube of the intubated anesthetized rat (70%nitrous oxide, 1.5% halothane, and 30% oxygen), and the injury wasinduced by the descent of a pendulum that strikes the piston. Ratsunderwent mild-to-moderate head injury, ranging from 1.6 to 1.9 atm.Brain temperature was indirectly monitored with a thermistor probeinserted into the right temporalis muscle and maintained at 37-37.5° C.Rectal temperature was also measured and maintained at 37° C. prior toand throughout the monitoring period.

II. Administration of Morphogen

[0214] Animals in the treatment group received OP1 intracistemally at adose of 10 μg/injection. Control animals received vehicle solutionslacking OP-1 but with all other components at equivalent finalconcentrations. Both OP-1 and vehicle-treated animals received twoinjections, one day and four days following the fluid percussion injury.

[0215] To administer the injection, the animals were anesthetized withhalothane in 70% NO₂/30% O₂ and placed in a stereotaxic frame. Theprocedure for intracistemal injection of OP1 containing solutions orvehicle-only solutions was identical. Using aseptic technique, OP-1 (1or 10 μg/injection ) or an equivalent volume of vehicle were introducedby percutaneous injection (10 μl/injection) into the cisterna magnausing a Hamilton syringe fitted with a 26 gauge needle (Yamada, et al.,(1991) J. Cereb. Blood Flow Metab. 11: 472-478). Before each injection,1-2 μl of cerebrospinal fluid (CSF) was drawn back through the Hamiltonsyringe to verify needle placement in the subarachnoid space.Preliminary studies demonstrated that a dye, 1% Evans blue, delivered inthis fashion diffused freely through the basal cisterns and over thecerebral cortex within one hour of injection. Animals were randomlyassigned to either of the OP-1 treatment groups or to the vehicletreatment group. Animals received two intracistemal injections (2×10μg/injection OP1 or 2× vehicle); the first injection was administered 24hours after the brain injury and the second injection was administered 4days after the brain injury.

III. Behavioral Testing

[0216] Three standard functional/behavioral tests were used to assesssensorimotor and reflex function after brain injury. The tests have beenfully described in the literature, including Bederson, et al., (1986)Stroke 17: 472-476; DeRyck, et al., (1992) Brain Res. 573: 44-60;Markgraf, et al., (1992) Brain Res. 575: 238-246; and Alexis, et al.,(1995) Stroke 26: 2338-2346.

A. The Forelimb Placing Test

[0217] Forelimb placing to three separate stimuli (visual, tactile, andproprioceptive) was measured to assess sensorimotor integration. DeRyck,et al., Brain Res. 573:44-60 (1992). For the visual placing subtest, theanimal is held upright by the researcher and brought close to a tabletop. Normal placing of the limb on the table is scored as “0,” delayedplacing (<2 sec) is scored as “1,” and no or very delayed placing (>2sec) is scored as “2.” Separate scores are obtained first as the animalis brought forward and then again as the animal is brought sideways tothe table (maximum score per limb=4; in each case higher numbers denotegreater deficits). For the tactile placing subtest, the animal is heldso that it cannot see or touch the table top with its whiskers. Thedorsal forepaw is touched lightly to the table top as the animal isfirst brought forward and then brought sideways to the table. Placingeach time is scored as above (maximum score per limb=4). For theproprioceptive placing subtest, the animal is brought forward only andgreater pressure is applied to the dorsal forepaw; placing is scored asabove (maximum score per limb=2). Finally, the ability of animals toplace the forelimb in response to whisker stimulation by the tabletopwas tested (maximum score per limb=2). Then subscores were added to givethe total forelimb placing score per limb (range=0-12).

B. The Beam Balance Test

[0218] Beam balance is sensitive to motor cortical insults. This taskwas used to assess gross vestibulomotor function by requiring a rat tobalance steadily on a narrow beam. Feeney, et al., Science, 217: 855-857(1982); Goldstein, et al., Behav. Neurosci. 104: 318-325 (1990). Thetest involved three 60-second training trials 24 hours before surgery toacquire baseline data. The apparatus consisted of a ¾-inch-wide beam, 10inches in length, suspended 1 ft. above a table top. The rat waspositioned on the beam and had to maintain steady posture with all limbson top of the beam for 60 seconds. The animals' performance was ratedwith the scale of Clifton, et al., J. Cereb Blood Flow Metab. 11:I114-121 (1991), which ranges from 1 to 6, with a score of 1 beingnormal and a score of 6 indicating that the animal was unable to supportitself on the beam.

C. The Beam Walking Test

[0219] This was a test of sensorimotor integration specificallyexamining hindlimb function. The testing apparatus and rating procedureswere adapted from Feeney, et al., Science, 217: 855-857 (1982). A1-inch-wide beam, 4 ft. in length, was suspended 3 ft. above the floorin a dimly lit room. At the far end of the beam was a darkened goal boxwith a narrow entryway. At equal distances along the beam, four 3-inchmetal screws were positioned, angling away from the beam's center. Awhite noise generator and bright light source at the start of the beammotivated the animal to traverse the beam and enter the goal box. Onceinside the goal box, the stimuli were terminated. The rat's latency toreach the goal box (in seconds) and hindlimb performance as it traversedthe beam (based on a 1 to 7 rating scale) were recorded. A score of 7indicates normal beam walking with less than 2 foot slips, and a scoreof 1 indicates that the rat was unable to traverse the beam in less than80 seconds. Each rat was trained for three days before surgery toacquire the task and to achieve normal performance (a score of 7) onthree consecutive trials. Three baseline trials were collected 24 hoursbefore surgery, and three testing trials were recorded daily thereafter.Mean values of latency and score for each day were computed.

IV. Results

[0220] As illustrated in FIGS. 16-18, OP-1 enhanced the recovery fromtraumatic brain injury in all three behavioral measures. In the forelimbplacing tests, OP-1-treated animals showed a gradual decrease in injuryseverity scores which attained statistical significance by day 9. SeeFIG. 16. In the beam balance and beam walking tests, OP-1-treatedanimals had performance scores that were essentially identical tosham-control animals. See FIGS. 17 and 18. These observations suggeststhat morphogens are capable of restoring impaired or lost sensory-motorfunctions following a traumatic brain injury, including visual, tactile,and proprioceptive placement, gross vestibulomotor function, andsensorimotor integration.

[0221] Similar routine modifications can be made in other acceptedmodels of traumatic central nervous system injury, to confirm efficacyof morphogen treatment to restore impaired or lost sensory-motorfunctions.

1 9 1822 base pairs nucleic acid single linear cDNA HOMO SAPIENSHIPPOCAMPUS CDS 49..1341 experimental /function= “OSTEOGENIC PROTEIN”/product=“OP1” /evidence=EXPERIMENTAL /standard_name= “OP1” 1 GGTGCGGGCCCGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57 Met His Val 1CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TTC GTG GCG CTC TGG GCA 105 ArgSer Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala 5 10 15 CCCCTG TTC CTG CTG CGC TCC GCC CTG GCC GAC TTC AGC CTG GAC AAC 153 Pro LeuPhe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn 20 25 30 35 GAGGTG CAC TCG AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG 201 Glu ValHis Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg 40 45 50 CGG GAGATG CAG CGC GAG ATC CTC TCC ATT TTG GGC TTG CCC CAC CGC 249 Arg Glu MetGln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg 55 60 65 CCG CGC CCGCAC CTC CAG GGC AAG CAC AAC TCG GCA CCC ATG TTC ATG 297 Pro Arg Pro HisLeu Gln Gly Lys His Asn Ser Ala Pro Met Phe Met 70 75 80 CTG GAC CTG TACAAC GCC ATG GCG GTG GAG GAG GGC GGC GGG CCC GGC 345 Leu Asp Leu Tyr AsnAla Met Ala Val Glu Glu Gly Gly Gly Pro Gly 85 90 95 GGC CAG GGC TTC TCCTAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC 393 Gly Gln Gly Phe Ser TyrPro Tyr Lys Ala Val Phe Ser Thr Gln Gly 100 105 110 115 CCC CCT CTG GCCAGC CTG CAA GAT AGC CAT TTC CTC ACC GAC GCC GAC 441 Pro Pro Leu Ala SerLeu Gln Asp Ser His Phe Leu Thr Asp Ala Asp 120 125 130 ATG GTC ATG AGCTTC GTC AAC CTC GTG GAA CAT GAC AAG GAA TTC TTC 489 Met Val Met Ser PheVal Asn Leu Val Glu His Asp Lys Glu Phe Phe 135 140 145 CAC CCA CGC TACCAC CAT CGA GAG TTC CGG TTT GAT CTT TCC AAG ATC 537 His Pro Arg Tyr HisHis Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile 150 155 160 CCA GAA GGG GAAGCT GTC ACG GCA GCC GAA TTC CGG ATC TAC AAG GAC 585 Pro Glu Gly Glu AlaVal Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp 165 170 175 TAC ATC CGG GAACGC TTC GAC AAT GAG ACG TTC CGG ATC AGC GTT TAT 633 Tyr Ile Arg Glu ArgPhe Asp Asn Glu Thr Phe Arg Ile Ser Val Tyr 180 185 190 195 CAG GTG CTCCAG GAG CAC TTG GGC AGG GAA TCG GAT CTC TTC CTG CTC 681 Gln Val Leu GlnGlu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu 200 205 210 GAC AGC CGTACC CTC TGG GCC TCG GAG GAG GGC TGG CTG GTG TTT GAC 729 Asp Ser Arg ThrLeu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp 215 220 225 ATC ACA GCCACC AGC AAC CAC TGG GTG GTC AAT CCG CGG CAC AAC CTG 777 Ile Thr Ala ThrSer Asn His Trp Val Val Asn Pro Arg His Asn Leu 230 235 240 GGC CTG CAGCTC TCG GTG GAG ACG CTG GAT GGG CAG AGC ATC AAC CCC 825 Gly Leu Gln LeuSer Val Glu Thr Leu Asp Gly Gln Ser Ile Asn Pro 245 250 255 AAG TTG GCGGGC CTG ATT GGG CGG CAC GGG CCC CAG AAC AAG CAG CCC 873 Lys Leu Ala GlyLeu Ile Gly Arg His Gly Pro Gln Asn Lys Gln Pro 260 265 270 275 TTC ATGGTG GCT TTC TTC AAG GCC ACG GAG GTC CAC TTC CGC AGC ATC 921 Phe Met ValAla Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser Ile 280 285 290 CGG TCCACG GGG AGC AAA CAG CGC AGC CAG AAC CGC TCC AAG ACG CCC 969 Arg Ser ThrGly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro 295 300 305 AAG AACCAG GAA GCC CTG CGG ATG GCC AAC GTG GCA GAG AAC AGC AGC 1017 Lys Asn GlnGlu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 310 315 320 AGC GACCAG AGG CAG GCC TGT AAG AAG CAC GAG CTG TAT GTC AGC TTC 1065 Ser Asp GlnArg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe 325 330 335 CGA GACCTG GGC TGG CAG GAC TGG ATC ATC GCG CCT GAA GGC TAC GCC 1113 Arg Asp LeuGly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala 340 345 350 355 GCCTAC TAC TGT GAG GGG GAG TGT GCC TTC CCT CTG AAC TCC TAC ATG 1161 Ala TyrTyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 360 365 370 AACGCC ACC AAC CAC GCC ATC GTG CAG ACG CTG GTC CAC TTC ATC AAC 1209 Asn AlaThr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn 375 380 385 CCGGAA ACG GTG CCC AAG CCC TGC TGT GCG CCC ACG CAG CTC AAT GCC 1257 Pro GluThr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala 390 395 400 ATCTCC GTC CTC TAC TTC GAT GAC AGC TCC AAC GTC ATC CTG AAG AAA 1305 Ile SerVal Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys 405 410 415 TACAGA AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCCTCC 1351 Tyr Arg AsnMet Val Val Arg Ala Cys Gly Cys His 420 425 430 GAGAATTCAG ACCCTTTGGGGCCAAGTTTT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411 GAACCAGCAG ACCAACTGCCTTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471 TGTGAGAGTA TTAGGAAACATGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531 ATCCAATGAA CAAGATCCTACAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591 GCATAAAGAA AAATGGCCGGGCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT 1651 CGTTTCCAGA GGTAATTATGAGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711 GGCGTGGCAA GGGGTGGGCACATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771 CTGTAATAAA TGTCACAATAAAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1822 431 amino acids amino acidlinear protein 2 Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser PheVal Ala 1 5 10 15 Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu AlaAsp Phe Ser 20 25 30 Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg ArgLeu Arg Ser 35 40 45 Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser IleLeu Gly Leu 50 55 60 Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His AsnSer Ala Pro 65 70 75 80 Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala ValGlu Glu Gly Gly 85 90 95 Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr LysAla Val Phe Ser 100 105 110 Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln AspSer His Phe Leu Thr 115 120 125 Asp Ala Asp Met Val Met Ser Phe Val AsnLeu Val Glu His Asp Lys 130 135 140 Glu Phe Phe His Pro Arg Tyr His HisArg Glu Phe Arg Phe Asp Leu 145 150 155 160 Ser Lys Ile Pro Glu Gly GluAla Val Thr Ala Ala Glu Phe Arg Ile 165 170 175 Tyr Lys Asp Tyr Ile ArgGlu Arg Phe Asp Asn Glu Thr Phe Arg Ile 180 185 190 Ser Val Tyr Gln ValLeu Gln Glu His Leu Gly Arg Glu Ser Asp Leu 195 200 205 Phe Leu Leu AspSer Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu 210 215 220 Val Phe AspIle Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg 225 230 235 240 HisAsn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser 245 250 255Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn 260 265270 Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe 275280 285 Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser290 295 300 Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val AlaGlu 305 310 315 320 Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys HisGlu Leu Tyr 325 330 335 Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp IleIle Ala Pro Glu 340 345 350 Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu CysAla Phe Pro Leu Asn 355 360 365 Ser Tyr Met Asn Ala Thr Asn His Ala IleVal Gln Thr Leu Val His 370 375 380 Phe Ile Asn Pro Glu Thr Val Pro LysPro Cys Cys Ala Pro Thr Gln 385 390 395 400 Leu Asn Ala Ile Ser Val LeuTyr Phe Asp Asp Ser Ser Asn Val Ile 405 410 415 Leu Lys Lys Tyr Arg AsnMet Val Val Arg Ala Cys Gly Cys His 420 425 430 102 amino acids aminoacid linear protein Protein 1..102 /label= OPX /note= “wherein each Xaais independently selected from a group of one or more specified aminoacids as defined in the specification.” 3 Cys Xaa Xaa His Glu Leu TyrVal Xaa Phe Xaa Asp Leu Gly Trp Xaa 1 5 10 15 Asp Trp Xaa Ile Ala ProXaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 20 25 30 Glu Cys Xaa Phe Pro LeuXaa Ser Xaa Met Asn Ala Thr Asn His Ala 35 40 45 Ile Xaa Gln Xaa Leu ValHis Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 50 55 60 Xaa Cys Cys Ala Pro ThrXaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 65 70 75 80 Asp Xaa Ser Xaa AsnVal Xaa Leu Xaa Lys Xaa Arg Asn Met Val Val 85 90 95 Xaa Ala Cys Gly CysHis 100 97 amino acids amino acid linear protein Protein 1..97 /label=Generic-Seq-7 /note= “wherein each Xaa is independently selected from agroup of one or more specified amino acids as defined in thespecification.” 4 Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly XaaCys Xaa Xaa Pro 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala XaaXaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys Cys Xaa Pro 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa XaaXaa Xaa Xaa Xaa 65 70 75 80 Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa ValXaa Xaa Cys Xaa Cys 85 90 95 Xaa 102 amino acids amino acid linearprotein Protein 1..102 /label= Generic-Seq-8 /note= “wherein each Xaa isindependently selected from a group of one or more specified amino acidsas defined in the specification.” 5 Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa XaaPhe Xaa Xaa Xaa Gly Trp Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Pro Xaa XaaXaa Xaa Ala Xaa Tyr Cys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa Pro Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Asn His Ala 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Cys Cys Xaa Pro Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Val XaaLeu Xaa Xaa Xaa Xaa Xaa Met Xaa Val 85 90 95 Xaa Xaa Cys Xaa Cys Xaa 10097 amino acids amino acid single linear protein Protein 1..97 /label=Generic-Seq-9 /note= “wherein each Xaa is independently selected from agroup of one or more specified amino acids as defined in thespecification.” 6 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly XaaCys Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Cys Xaa Pro 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa XaaXaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Cys Xaa Cys 85 90 95 Xaa 102 amino acids amino acid singlelinear protein Protein 1..102 /label= Generic-Seq-10 /note= “whereineach Xaa is independently selected from a group of one or more specifiedamino acids as defined in the specification.” 7 Cys Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa XaaPro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Cys Xaa ProXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Cys XaaCys Xaa 100 5 amino acids amino acid linear peptide Protein 1..5 /note=“wherein each Xaa is independently selected from a group of one or morespecified amino acids as defined in the specification” 8 Cys Xaa Xaa XaaXaa 1 5 5 amino acids amino acid linear peptide Protein 1..5 /note=“wherein each Xaa is independently selected from a group of one or morespecified amino acids as defined in the specification” 9 Cys Xaa Xaa XaaXaa 1 5

What is claimed is:
 1. A method of treating amyotrophic lateral sclerosis, comprising administering a morphogen comprising a dimeric protein having an amino acid sequence selected from the group consisting of a sequence: (a) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO:2; (b) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO:4; (d) defined by Generic Sequence 8, SEQ ID NO:5; (e) defined by Generic Sequence 9, SEQ ID NO:6; (f) defined by Generic Sequence 10, SEQ ID NO:7, and (g) defined by OPX, SEQ ID NO:3, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 2. A method of treating multiple sclerosis, comprising administering a morphogen comprising a dimeric protein having an amino acid sequence selected from the group consisting of a sequence: (a) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO:2; (b) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO:4; (d) defined by Generic Sequence 8, SEQ ID NO:5; (e) defined by Generic Sequence 9, SEQ ID NO:6; (f) defined by Generic Sequence 10, SEQ ID NO:7, and (g) defined by OPX, SEQ ID NO:3, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 3. A method of treating a spinal cord injury, comprising administering a morphogen comprising a dimeric protein having an amino acid sequence selected from the group consisting of a sequence: (a) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO:2; (b) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO:4; (d) defined by Generic Sequence 8, SEQ ID NO:5; (e) defined by Generic Sequence 9, SEQ ID NO:6; (f) defined by Generic Sequence 10, SEQ ID NO:7, and (g) defined by OPX, SEQ ID NO:3, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 4. The method of claim 3, wherein said spinal cord injury results from a mechanical trauma.
 5. The method of claim 3, wherein said spinal cord injury results from a tumor.
 6. The method of claim 3, wherein said spinal cord injury results from a chemical trauma.
 7. A method of restoring motor function in a mammal afflicted with amyotrophic lateral sclerosis, comprising administering a morphogen comprising a dimeric protein having an amino acid sequence selected from the group consisting of a sequence: (a) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO:2; (b) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO:4; (d) defined by Generic Sequence 8, SEQ ID NO:5; (e) defined by Generic Sequence 9, SEQ ID NO:6; (f) defined by Generic Sequence 10, SEQ ID NO:7, and (g) defined by OPX, SEQ ID NO:3, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 8. A method of restoring motor function in a mammal afflicted with multiple sclerosis, comprising administering a morphogen comprising a dimeric protein having an amino acid sequence selected from the group consisting of a sequence: (a) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO:2; (b) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO:4; (d) defined by Generic Sequence 8, SEQ ID NO:5; (e) defined by Generic Sequence 9, SEQ ID NO:6; (f) defined by Generic Sequence 10, SEQ ID NO:7, and (g) defined by OPX, SEQ ID NO:3, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 9. A method of restoring motor function in a mammal afflicted with a spinal cord injury, comprising administering a morphogen comprising a dimeric protein having an amino acid sequence selected from the group consisting of a sequence: (a) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO:2; (b) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO:4; (d) defined by Generic Sequence 8, SEQ ID NO:5; (e) defined by Generic Sequence 9, SEQ ID NO:6; (f) defined by Generic Sequence 10, SEQ ID NO:7, and (g) defined by OPX, SEQ ID NO:3, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 10. A method of preserving motor function in a mammal afflicted with or at risk of amyotrophic lateral sclerosis, comprising administering a morphogen comprising a dimeric protein having an amino acid sequence selected from the group consisting of a sequence: (a) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO:2; (b) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO:4; (d) defined by Generic Sequence 8, SEQ ID NO:5; (e) defined by Generic Sequence 9, SEQ ID NO:6; (f) defined by Generic Sequence 10, SEQ ID NO:7, and (g) defined by OPX, SEQ ID NO:3, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 11. A method of preserving motor function in a mammal afflicted with or at risk of multiple sclerosis, comprising administering a morphogen comprising a dimeric protein having an amino acid sequence selected from the group consisting of a sequence: (a) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO:2; (b) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO:4; (d) defined by Generic Sequence 8, SEQ ID NO:5; (e) defined by Generic Sequence 9, SEQ ID NO:6; (f) defined by Generic Sequence 10, SEQ ID NO:7, and (g) defined by OPX, SEQ ID NO:3, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 12. A method of preserving motor function in a mammal afflicted with or at risk of a spinal cord injury, comprising administering a morphogen comprising a dimeric protein having an amino acid sequence selected from the group consisting of a sequence: (a) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1, residues 330-431 of SEQ ID NO:2; (b) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO:4; (d) defined by Generic Sequence 8, SEQ ID NO:5; (e) defined by Generic Sequence 9, SEQ ID NO:6; (f) defined by Generic Sequence 10, SEQ ID NO:7, and (g) defined by OPX, SEQ ID NO:3, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 13. A method of treating amyotrophic lateral sclerosis, comprising administering a morphogen selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vg1, Vgr-1, BMP3, BMP5, and BMP6, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 14. A method of treating multiple sclerosis, comprising administering a morphogen selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vg1, Vgr-1, BMP3, BMP5, and BMP6, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 15. A method of treating a spinal cord injury, comprising administering a morphogen selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vg1, Vgr-1, BMP3, BMP5, and BMP6, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 16. A method of restoring motor function in a mammal afflicted with amyotrophic lateral sclerosis, comprising administering a morphogen selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vg1, Vgr-1, BMP3, BMP5, and BMP6, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 17. A method of restoring motor function in a mammal afflicted with multiple sclerosis, comprising the step of administering a morphogen selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vg1, Vgr-1, BMP3, BMP5, and BMP6, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 18. A method of restoring motor function in a mammal afflicted with a spinal cord injury, comprising administering a morphogen selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vg1, Vgr-1, BMP3, BMP5, and BMP6, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 19. A method of preserving motor function in a mammal afflicted with or at risk of amyotrophic lateral sclerosis, comprising administering a morphogen selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vg1, Vgr-1, BMP3, BMP5, and BMP6, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 20. A method of preserving motor function in a mammal afflicted with or at risk of multiple sclerosis, comprising administering a morphogen selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vg1, Vgr-1, BMP3, BMP5, and BMP6, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 21. A method of preserving motor function in a mammal afflicted with or at risk of a spinal cord injury, comprising administering a morphogen selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vg1, Vgr-1, BMP3, BMP5, and BMP6, wherein said morphogen stimulates production of an N-CAM or L1 isoform by an NG108-15 cell in vitro.
 22. The method of claim 1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, wherein said morphogen is complexed with at least one pro-domain polypeptide selected from the group consisting of the pro-domains of OP-1, OP-2, 60A, GDF-1, BMP-2A, BMP-2B, DPP, Vg1, Vgr-1, BMP-3, BMP-5, and BMP-6.
 23. The method of claim 22, wherein said morphogen is complexed with a pair of said pro-domain polypeptides. 